The role of 5-HT1A and 5-HT1B receptors in MDMA self ...

The role of 5-HT1A and 5-HT1B receptors in MDMA self-administration

By

Dane Aronsen

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

Victoria University of Wellington

2016

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Acknowledgements

To everyone that made this 3 or so years such a positive experience, thank you. I

couldn’t have done it without the family and friends that supported me through thick

and thin.

I want to express sincere gratitude to the team of people I worked with in the Lab, with

a special shout out to Joyce for patiently teaching me whatever I needed to know, and

my partner in all lab shenanigans, Jeremy.

A big thank you to the academic staff that presented me with challenges and

opportunities, and who would happily write me a scholarship reference at the last

minute.

And Sue. You’ve taught me a lot, and some of it was about science. You’ve made sure

I will never forget my experience as a PhD student.

Thanks guys.

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Contents

Acknowledgements ......................................................................................................... 3  

List of abbreviations ........................................................................................................ 6  

List of ligands .................................................................................................................. 7  

Abstract ........................................................................................................................... 8  

General Introduction ..................................................................................................... 10  

Brief history of MDMA ............................................................................................ 10  

MDMA use ............................................................................................................... 11  

Harms associated with MDMA use .......................................................................... 13  

Problems associated with studying the harmful effects of MDMA .......................... 13  

Animal studies ........................................................................................................... 15  

Profile of MDMA self-administration ...................................................................... 18  

Pharmacodynamics of MDMA ................................................................................. 23  

A focus on 5-HT ....................................................................................................... 24  

5-HT1A and 5-HT1B receptors .................................................................................... 26  

Summary ................................................................................................................... 35  

General Methods ........................................................................................................... 37  

Subjects ..................................................................................................................... 37  

Drugs ......................................................................................................................... 37  

Apparatus and procedures ......................................................................................... 37  

Chapter 3: Development of behavioural assays ............................................................ 39  

Method ...................................................................................................................... 41  

Results ....................................................................................................................... 42  

Discussion ................................................................................................................. 43  

Chapter 4: Behavioural responses to RU 24969 ........................................................... 45  

Method ...................................................................................................................... 46  

Results ....................................................................................................................... 48  

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Discussion .................................................................................................................. 51  

Chapter 5: Effects of repeated administration of the 5-HT1B/1A receptor agonist, RU

24969, on the acquisition of MDMA self-administration ............................................. 54  

Method ....................................................................................................................... 56  

Results ....................................................................................................................... 58  

Discussion .................................................................................................................. 61  

Chapter 6: Predicting the acquisition of MDMA self-administration ........................... 66  

Method ....................................................................................................................... 67  

Results ....................................................................................................................... 69  

Discussion .................................................................................................................. 72  

Chapter 7: Response to 5-HT1A and 5-HT1B receptor agonists after

self-administration ........................................................................................................ 75  

Method ....................................................................................................................... 78  

Results ....................................................................................................................... 79  

Discussion .................................................................................................................. 82  

General Discussion ........................................................................................................ 84  

Summary .................................................................................................................... 84  

Synthesis of results .................................................................................................... 85  

Limitations ................................................................................................................. 86  

Validity of MDMA doses .......................................................................................... 87  

Key findings and future directions ............................................................................ 89  

Conclusion ................................................................................................................. 93  

References ..................................................................................................................... 95  

Appendix A: Publication details and permissions ....................................................... 126  

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List of abbreviations

5,7-DHT: 5,7-Dihydroxytryptamine

5CSRTT: 5 choice serial reaction time task

5-HIAA: 5-Hydroxyindoleacetic acid

5-HT: 5-hydroxytryptamine; serotonin

6-OH-DA 6-hydroxydopamine

CSF: Cerebrospinal fluid

DSM: Diagnostic and Statistical Manual of Mental Disorders

EC50: Half maximal effective concentration

EPM: Elevated plus maze

FR: Fixed ratio

GABA: gamma-Aminobutyric acid

GTPγS: guanosine 5'-O-[gamma-thio]triphosphate

Ki: Binding affinity (amount of ligand required to bind 50% of receptors)

MDMA : 3,4-methylenedioxymethamphetamine

NAc: Nucleus accumbens

PFC: Prefrontal cortex

SSRI: Selective serotonin reuptake inhibitor

SUD: Substance use disorder

VTA: Ventral tegmental area

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List of ligands

5-MeODMT 5-HT2/1A receptor agonist

8-OH-DPAT 5-HT1A/7 receptor agonist

BAY × 3702 5-HT1A receptor agonist

CGS 12066 5-HT1B/2 receptor agonist

CP 93129 5-HT1B receptor agonist

CP 94253 5-HT1B receptor agonist

DOI 5-HT2A/2C receptor agonist

F13640 5-HT1A receptor agonist

F15599 5-HT1A receptor agonist

Fluoxetine Serotonin uptake inhibitor

GBR 12909 Dopamine uptake inhibitor

GR 127935 5-HT1B/1D receptor antagonist

Ketanserin 5-HT2A/2C receptor and H1 receptor antagonist

M100907 5-HT2A receptor antagonist

mCPP 5-HT2/1A receptor agonist

MK 212 5-HT2C receptor agonist

Pindolol β1- and β2-adrenergic receptor antagonist, 5-HT1A antagonist

Propanolol β1- and β2-adrenergic receptor antagonist, 5-HT1A/1B antagonist

Ro 60-175 5-HT2C receptor agonist

RU 24969 5-HT1B/1A receptor agonist

SB 206553 5-HT2C/2B receptor antagonist

SB 224289 5-HT1B receptor inverse agonist

SB 242084 5-HT2C receptor antagonist

SDX 216-525 5-HT1A receptor antagonist

Tianeptine 5-HT uptake facilitator

WAY 101405 5-HT1A receptor antagonist

WAY 100635 5-HT1A receptor antagonist

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Abstract

Rationale: 3,4-methylenedioxymethamphetamine (MDMA) is a less efficacious

reinforcer than other drugs of abuse. However, following repeated self-administration,

responding increases for some animals and efficacy becomes comparable to other

drugs of abuse. MDMA-stimulated serotonin (5-HT) release was negatively associated

with acquisition of MDMA self-administration, and a neurotoxic 5-HT lesion reduced

the latency to acquire self-administration. These findings suggest that MDMA-

produced 5-HT release is an important component of self-administration. The receptor

mechanisms are not, however, well understood, although it has often been suggested

that the mechanism involves 5-HT-mediated inhibition of dopamine. Both 5-HT1A and

5-HT1B receptors are well localised to regulate dopamine release, and both have been

implicated in modulating the reinforcing effects of many drugs of abuse.

Objectives: The first objective was to establish specific behavioural assays to reflect

5-HT1A and 5-HT1B receptor activation. Then, using the established behavioural

assays, the aim was to determine the role of 5-HT1A and 5-HT1B receptors in the

acquisition of MDMA self-administration. The impact of substantial MDMA self-

administration on 5-HT1A and 5-HT1B receptors was also assessed.

Methods: Firstly, dose-effect relationships for the hyperactive response to the 5-HT1A

receptor agonist, 8-OH-DPAT (0 – 3.0 mg/kg) and the hyperactive and adipsic

response to the 5-HT1B/1A receptor agonist, RU 24969 (0 – 3.0 mg/kg) were

determined. Selectivity of these responses was determined by co-administration of the

5-HT1A receptor antagonist, WAY 100635, or the 5-HT1B/1D receptor antagonist, GR

127935. Secondly, a pretreatment regimen of the RU 24969 (2 × 3.0 mg/kg/day, 3

days), which had been suggested to down-regulate 5-HT1B/1A receptors, was

administered prior to self-administration testing. The effect of this manipulation on

both the acquisition of MDMA self-administration, and the behavioural responses to 5-

HT1A and 5-HT1B receptor activation, was measured. A further study measured

behavioural responses to 5-HT1A or 5-HT1B receptor agonists prior to self-

administration, to determine whether the variability in these responses would predict

the variability in the latency to acquisition of MDMA self-administration. Lastly, the

effect of substantial MDMA self-administration (350 mg/kg) on dose-response curves

for the behavioural effects of 5-HT1A or 5-HT1B receptor activation was assessed.

Results: The hyperactive response to the 5-HT1B/1A receptor agonist, RU 24969, was

blocked by the 5-HT1A receptor antagonist, WAY 100635, but not the 5-HT1B receptor

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antagonist, GR127935. Similarly, the hyperactive response to the 5-HT1A receptor

agonist, 8-OH-DPAT, was dose-dependently blocked by WAY 100635. GR 127935,

but not WAY 100635, blocked the adipsic response to RU 24969.

Repeated administration of RU 24969 produced rightward shifts in the dose-response

curves for 8-OH-DPAT-produced hyperactivity and RU 24969-produced adipsia, and

also greatly facilitated the acquisition of MDMA self-administration. However, there

was no correlation between latency to acquire MDMA self-administration and the

hyperactive response to 8-OH-DPAT or the adipsic response to RU 24969, and

MDMA self-administration failed to alter these behavioural response to activation of

5-HT1A or 5-HT1B receptors.

Conclusions: The hyperactive response to 8-OH-DPAT and the adipsic response to

RU 24969 reflect activation of 5-HT1A and 5-HT1B receptors, respectively. The

variability in acquisition of MDMA self-administration was reduced by a treatment

that also down-regulated 5-HT1A and 5-HT1B receptors, however there was no further

indication that these receptors play a critical role in the self-administration of MDMA.

Instead, it seems likely that other 5-HT receptors have a greater impact on MDMA

self-administration.

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General Introduction

Parts of this chapter have been adapted from:

Aronsen & Schenk (2016). MDMA self-administration fails to alter the behavioral

response to 5-HT1A and 5-HT1B agonists. Psychopharmacology, 233 (7), 1323-

1330. DOI 10.1007/s00213-016-4226-9

Aronsen, Bukholt, & Schenk (2016). Repeated administration of the 5-HT1B/1A agonist,

RU 24969, facilitates the acquisition of MDMA self-administration: Role of 5-

HT1A and 5-HT1B receptor mechanisms. Psychopharmacology, 233 (8), 1339-

1347. DOI 10.1007/s00213-016-4225-x

Schenk & Aronsen (2015). Contribution of Impulsivity and Serotonin Receptor

Neuroadaptations to the Development of an MDMA (“ecstasy”) Substance Use

Disorder, in Current Topics in Behavioral Neuroscience: The Misuse of Licit

and Illicit Drugs in Psychopharmacology Susan Schenk, Suzanne Nielson,

Raimondo Bruno (Eds), Springer. DOI 10.1007/7854_2015_421

Aronsen, Webster, & Schenk (2014). RU 24969-produced adipsia and

hyperlocomotion: Differential role of 5HT1A and 5HT1B receptor mechanisms.

Pharmacology, Biochemistry and Behavior, 124, 1-4. DOI

10.1016/j.pbb.2014.05.008

with permission from the publisher (Appendix A).

Brief history of MDMA

3,4-methylenedioxymethamphetamine (MDMA) was initially patented by the

pharmaceutical company, Merck, in 1914 as a precursor for other therapeutically

efficacious compounds (Green, Mechan, Elliott, O'Shea, & Colado, 2003). Some basic

preclinical tests were conducted with MDMA in 1927 (Freudenmann, Öxler, &

Bernschneider-­‐Reif, 2006), and in 1953 the US Army sponsored research on the

toxicity of MDMA, concluding that further study in humans should be conducted

(Hardman, Haavik, & Seevers, 1973). In response to the first reports of recreational

MDMA use (Gaston & Rasmussen, 1972) Alexander Shulgin published the first papers

outlining the effects of MDMA in humans (Anderson, Braun, Braun, Nichols, &

Shulgin, 1978; Shulgin, 1978; Shulgin & Nichols, 1978), also encouraging further

human studies. Shulgin was a vocal advocate for the use of MDMA as an adjunct to

psychotherapy, but it has been suggested that his public promotion of MDMA also led

to increased recreational use (Benzenhöfer & Passie, 2010).

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As recreational use continued to grow, there was pressure on governments to

bring the use of MDMA under legislative control (Beck & Rosenbaum, 1990). MDMA

was scheduled as a Class B Controlled Drug in New Zealand in 1987 (New Zealand

Drug Foundation, 2015), following classification in Schedule I by the USA Drug

Enforcement Agency in 1985 (Beck & Rosenbaum, 1990). The import, manufacture,

supply, or administration of Class B Controlled Drugs carries a jail sentence of up to

14 years in New Zealand (Misuse of Drugs Act 1975). Nonetheless, recreational use of

MDMA, in the form of the street drug, ‘ecstasy’, is popular in New Zealand (Wilkins,

2011; Wilkins & Sweetsur, 2008), and around the world (United Nations Office on

Drugs and Crime, 2015).

Recently, there has been a revival in the push to harness the subjective effects

of MDMA in the treatment of psychiatric disorders. Some therapists claim that

MDMA helps patients talk openly, and fosters an atmosphere of trust (Kupferschmidt,

2014). Clinical trials are currently underway assessing the utility of MDMA as a

therapeutic adjunct in the treatment of post-traumatic stress disorder, and anxiety

associated with a life-threatening illness (National Institutes of Health, 2015).

MDMA use

MDMA is generally consumed as the primary psychoactive component of the

popular street drug, ecstasy (also known as E, Molly, pingers, pills, disco biscuits).

Ecstasy is most commonly available in tablet form, and tablets are usually either

consumed orally or crushed for intranasal administration (De La Garza, Fabrizio, &

Gupta, 2007; Parrott, 2013a; Solowij, Hall, & Lee, 1992). In recent years

recreationally used ecstasy tablets have been shown to contain a wide range of

psychoactive substances, including significant quantities of methamphetamine,

ketamine, caffeine, meta-Chlorophenylpiperazine (mCPP) and mephedrone, and have

sometimes contained no MDMA whatsoever (Brunt, Koeter, Niesink, & van den

Brink, 2012; Morefield, Keane, Felgate, White, & Irvine, 2011; Togni, Lanaro,

Resende, & Costa, 2015; Vogels et al., 2009). Therefore, throughout this thesis, the

term ‘ecstasy’ will be used to refer to the street drug that generally contains MDMA,

while ‘MDMA’ will be used to refer specifically to the psychoactive substance.

Ecstasy became popular in the underground dance party scene of the 1980s, in

part because it increases energy levels, heightens sensual awareness, and facilitates

bonding (McDowell & Kleber, 1994; Schwartz & Miller, 1997). In the 1990s and early

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2000s ecstasy use became more mainstream, becoming a popular recreational drug

among young adults. A recent study reported worldwide prevalence of ecstasy use to

be the second highest of all illicit drugs (Global Drug Survey, 2014). Recently,

popularity of ecstasy has been facilitated by a ‘re-branding’ of ecstasy as ‘Molly’ in

the mainstream media. ‘Ecstasy’ has associations with the old dance parties of the

1980s, electronic music, and un-masculine displays of affection, misaligning it with

the modern zeitgeist which is heavily influenced by pop and hip-hop culture. On the

other hand, ‘Molly’ has been embraced by the hip-hop and pop communities,

providing a ‘new’ drug that youth can associate with (Carter, 2016).

Although ecstasy use is common, patterns of use differ widely. A recent study

showed that, of 109 subjects who had recently used ecstasy for the first time, 43 did

not take ecstasy again in the following 12 months, while 23 consumed more than 10

ecstasy pills in that time period (Wagner, Becker, Koester, Gouzoulis-­‐Mayfrank, &

Daumann, 2013), illustrating that some will use ecstasy very infrequently, while others

will use ecstasy regularly. Furthermore, recent surveys have found a significant

proportion of regular ecstasy users met Diagnostic and Statistical Manual of Mental

Disorders (DSM) -IV-based criteria for dependence (Cottler, Leung, & Abdallah,

2009; Cottler, Womack, Compton, & Ben-Abdallah, 2001; Uosukainen, Tacke, &

Winstock, 2015). The more recent DSM 5 provides diagnostic criteria for ‘substance

use disorders’ (SUDs) rather than ‘dependence’ (American Psychiatric Association,

2013). Although there is no specific ecstasy SUD, some ecstasy users met a number of

SUD criteria, including using more drug than intended (Cottler et al., 2009; Cottler et

al., 2001) unsuccessful efforts to cut down on use (Jansen, 1999), craving (A. K. Davis

& Rosenberg, 2014; Hopper et al., 2006), neglecting activities other than acquiring and

taking drug (Cottler et al., 2009; Cottler et al., 2001; Jansen, 1999; Yen & Hsu, 2007),

use in spite of known negative consequences (Cottler et al., 2009; Cottler et al., 2001;

Jansen, 1999; Schifano & Magni, 1994; Yen & Hsu, 2007), tolerance (Cottler et al.,

2001; Jansen, 1999; Kirkpatrick et al., 2014; Parrott, 2005; Peroutka, Newman, &

Harris, 1988; Yen & Hsu, 2007), and withdrawal (Cottler et al., 2009; Cottler et al.,

2001; Jansen, 1999; Peroutka et al., 1988). Thus, while some ecstasy users take ecstasy

relatively infrequently, a subpopulation of users show regular use, and some show

signs of an SUD.

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Harms associated with MDMA use

The regular use of ecstasy in some users is of concern, not only because of the

potential to develop an SUD, but also because ecstasy use has been associated with a

number of cognitive, behavioural, and neurochemical deficits. Ecstasy users showed

deficits in learning (Wagner et al., 2013), and in attention and memory (McCann,

Mertl, Eligulashvili, & Ricaurte, 1999) compared to ecstasy-naïve controls or those

with limited ecstasy use. Ecstasy users reported higher levels of depression,

impulsiveness, and sleep disturbances than poly-drug users who did not use ecstasy

(Soar, Turner, & Parrott, 2006; Taurah, Chandler, & Sanders, 2014). These cognitive

and behavioural deficits were persistent, suggesting that regular ecstasy use may cause

long-lasting neuroadaptations (Parrott, 2013a, 2013b; Schifano & Magni, 1994). With

increased experience some heavy ecstasy users report persistent problematic

behaviour, including paranoid delusions (Schifano & Magni, 1994), severe weight loss

(Jansen, 1999; Schifano & Magni, 1994), and suicidal thought (Jansen, 1999; Schifano

& Magni, 1994).

Ecstasy use has also been associated with deficits in the neurotransmitter,

serotonin (5-HT). Ecstasy users had decreased 5-HT transporter binding (Kish et al.,

2010; McCann, Szabo, Scheffel, Dannals, & Ricaurte, 1998), reduced levels of the 5-

HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in cerebrospinal fluid (McCann,

Mertl, et al., 1999), reduced 5-HT synthesis in frontal and parietal regions (Booij et al.,

2014), and autopsied striatal tissue from a heavy ecstasy user indicated decreased 5-

HT and 5-HIAA levels (Kish, Furukawa, Ang, Vorce, & Kalasinsky, 2000). These

markers of reduced 5-HT function correlate with lifetime ecstasy use (Kish et al.,

2010; McCann et al., 1998) and levels of behavioural impairment (Kish et al., 2010).

Therefore, it is possible that at least some of these adaptations underlie the long term

behavioural problems seen after repeated ecstasy use. The mechanisms by which

MDMA might produce these effects is not clear.

Problems associated with studying the harmful effects of MDMA

Given the global popularity of ecstasy, and the deficits associated with regular

use, it becomes important to investigate potential treatments to reduce intake, and to

reverse harmful neuroadaptations. However, there are a number of potential confounds

associated with studies that use human subjects to determine the effects of MDMA use

on the brain and/or behaviour. Firstly, results from studies on ecstasy users can be

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limited by a number of factors. For example, the accuracy of subjects’ reported use and

the range of other drugs the subject also uses may confound results. This concept is

illustrated in the abovementioned report by Jansen (1999) describing the effects of

ecstasy use in three regular users, in which total lifetime ecstasy exposure was

determined by self-report for periods of over two years. This method for determining

drug intake relies on memory for drug taking episodes even though ecstasy use is

associated with memory impairments. Furthermore, the study by Jansen highlights the

poly-drug use typical of regular ecstasy users (Cottler et al., 2009; Cottler et al., 2001)

– the first patient reported regular amphetamine use of 1g/day, the second was

dependent on benzodiazepines, while the third consumed roughly 1 bottle of spirits

every night. Regular use of other drugs makes it more difficult to isolate the effects of

MDMA.

A second potential issue with human studies is that varied individual histories

of ecstasy users can limit the conclusions that can be drawn. For example, while

symptoms of depression and anxiety are widely reported after regular ecstasy use

(Rogers et al., 2009), a causal link cannot be drawn between ecstasy use and

psychological deficits, given that pre-existing problems such as anxiety and depression

might predispose an individual to regular ecstasy consumption as a form of self-

medication (Parrott, 2006, 2013a). Without random allocation and an appropriate

control group, causal links between drug use and its effects cannot be drawn. In an

effort to overcome this limitation a small number of studies have randomly assigned

participants to receive acute administrations of MDMA, but, as the authors of one

study lament, ethical constraints on dosing regimens severely limit the ecological

validity and scope of such studies (Peiró et al., 2013).

Some researchers have gone to great lengths to minimise the impact of such

confounds on the results of their studies. For example, a recent study investigated

current ecstasy users and compared results to a control group of poly-drug users that

have never used ecstasy. Thus, any differences should be attributable to ecstasy use.

The results showed that ecstasy users had higher levels of cognitive and behavioural

disturbances than non-ecstasy poly-drug users (Taurah et al., 2014). Such results

strengthen claims that MDMA use is harmful and help to illustrate the nature of these

harms. However, because of ethical constraints that restrict the doses of MDMA that

can be administered to humans, investigations into the mechanisms behind these

effects of MDMA cannot be readily conducted.

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Animal studies

For these reasons, animal models are often turned to in order to obtain

information regarding the effects of exposure to MDMA. The real value of animal

laboratory studies is that they allow experimenters some control over the histories of

subjects, the drugs administered, and environmental factors. Furthermore, a wider

range of doses can be administered to animals than is ethically viable with humans.

There is some loss of ecological validity when animal models are employed,

particularly as they necessarily ignore the complex environment in which ecstasy is

consumed, but such studies can be incredibly helpful in evaluating properties of

MDMA that cannot be determined in humans.

A number of studies have replicated the findings of human studies after

administering MDMA to animals. Typically, high doses of MDMA are administered

repeatedly, after which some behavioural or neurochemical measures are made. For

example, exposure to high doses of experimenter-administered MDMA decreased

tissue 5-HT levels (Battaglia, Yeh, & De Souza, 1988; Commins et al., 1987;

McGregor et al., 2003), damaged 5-HT cells (Commins et al., 1987; Jensen et al.,

1993), and reduced 5-HT transporter binding (Battaglia, Yeh, et al., 1988; McGregor

et al., 2003). In behavioural tests, repeated administration of MDMA increased

anxiety-like behaviour in adult (McGregor et al., 2003) and adolescent rats (Bull,

Hutson, & Fone, 2003; Bull, Hutson, & Fone, 2004; Cox et al., 2014), and impaired

novel object discrimination, a measure of recognition memory (Shortall et al., 2013).

Although this method of experimenter-administered, high dose MDMA is useful for

determining the harmful effects of MDMA, these studies have been criticised for

employing a physiologically irrelevant dosing regimen (Baumann & Rothman, 2009;

Cole & Sumnall, 2003; De La Garza et al., 2007; Meyer, Piper, & Vancollie, 2008),

given that this high level of exposure is rarely, if ever, experienced by ecstasy users

(D. Hansen, Maycock, & Lower, 2001; Parrott, 2005; Verheyden, Henry, & Curran,

2003).

One alternative to an experimenter administered drug regimen is to give the

animal control over the delivery of drug, in a manner similar to how humans control

their drug intake. This is the basis of the popular self-administration paradigm, in

which an animal performs some operant (e.g. nose poke, lever press) in order to obtain

a dose of drug. Often, the route of drug administration is intravenous, meaning the

animal requires a surgically implanted indwelling venous catheter. After recovery from

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this surgery, the animal is placed in an operant chamber and the catheter is connected

via tubing to a syringe encased in a mechanical syringe pump. The operant activates

the syringe pump, resulting in a predetermined intravenous dose of the drug being

investigated. Drug infusions are generally paired with a stimulus (e.g. light, tone).

Usually there is a second manipulandum (e.g. nose poke hole, lever) for which the

operant has no programmed consequence, but responses are recorded as a measure of

non-specific responding.

Human drug taking is a complex behaviour that is influenced by an interaction

of social, economic, and personal factors, and as such it cannot be modelled in a single

animal paradigm. Furthermore, as with all animal models, ecological validity is lost in

order to gain experimental control and practicality. For example, self-administration

studies generally allow an animal to self-administer only the drug of interest (with no

adulterants), in order to draw causal conclusions about this drug. In contrast, human

drug users tend to use a range of drugs, and drugs procured on the street tend not to be

pure. Thus, the self-administration paradigm trades ecological validity for

experimental control (De La Garza et al., 2007). While experimental design can help to

minimise the loss of validity, no self-administration model can perfectly replicate

human drug taking. Nonetheless, as will be explained below, the self-administration

paradigm is an excellent paradigm for MDMA administration, and also allows for

studies in which drug taking is the dependent measure.

A particular strength of the self-administration paradigm is that the animal has

control over their drug intake. Firstly, this reduces concerns over the administration of

irrelevantly large drug doses. Figure 1.1 presents data adapted from Schenk, Gittings,

Johnstone, and Daniela (2003) showing the number of infusions of MDMA that were

self-administered in a session, for different doses of MDMA. It is clear that MDMA

self-administration behaviour adjusts as dose changes, illustrating that the animal

utilises control over responding to regulate total drug intake. Thus, it is less likely that

physiologically irrelevant doses will be administered, as has been suggested for studies

using experimenter-administered MDMA.

17

M D M A (m g /k g /in fu s io n )

Infu

sio

ns

pe

r 2

hr

se

ss

ion

0

2 0

4 0

6 0

8 0

1 0 0

0 .25 0 .5 1 .0 2 .0

Fig. 1.1 Dose  response  curve  for  MDMA  self-­‐administration.  Adapted  from  Schenk  et  al.  (2003).  

Secondly, self-administered drug produces neuroadaptations that are not solely

due to the action of the drug. For example, self-administered cocaine produced

significantly greater changes in dopamine transporter binding than the same doses

administered non-contingently, suggesting that the stimulus-response associations

learned in self-administration contribute to the neuroadaptations produced by drugs of

abuse (Miguéns et al., 2008). Because human users also have control over their drug

intake, and because the neuroadaptations produced by drugs may be dependent on this

control, self-administered MDMA is probably a better model of human drug

administration than experimenter-administered MDMA.

A third strength of the self-administration paradigm, and of particular relevance

to MDMA, is that the overall pattern of drug taking is similar in animals and humans.

On their first exposure to MDMA human users generally consume ½ - 1 ecstasy tablet

(D. Hansen et al., 2001) with drug use being intermittent, but with experience some

users may consume upwards of 20 pills in a session (Parrott, 2005; Verheyden et al.,

2003). A similar pattern of low, intermittent initial intake followed by increased intake

in some subjects is seen in MDMA self-administration in rats and monkeys (Banks et

al., 2008; Beardsley, Balster, & Harris, 1986; De La Garza et al., 2007; Schenk,

Colussi-Mas, Do, & Bird, 2012). It is important that, in both animals and humans,

initial exposure to MDMA is low and intermittent, because intermittent or low dose

18

exposure to MDMA was neuroprotective against the neuroadaptations produced by

subsequent high dose administrations (Bhide, Lipton, Cunningham, Yamamoto, &

Gudelsky, 2009; Piper, Ali, Daniels, & Meyer, 2010). Indeed, self-administered

MDMA produced smaller deficits in tissue levels of 5-HT compared to high dose

experimenter-administered MDMA (Do & Schenk, 2011; Scanzello, Hatzidimitriou,

Martello, Katz, & Ricaurte, 1993; Schenk et al., 2007), even though the total amount

self-administered (165-350 mg/kg over 20-30 days of testing) was greater than is

generally administered to produce extensive neurotoxicity (20-80 mg/kg in a single

day). Given that the neuroadaptations produced by MDMA are dependent on the

pattern of prior MDMA exposure, self-administered MDMA likely produces

neuroadaptations more similar to the human condition than those produced by

experimenter-administration.

Furthermore, the self-administration paradigm allows for the behaviour of drug

taking to be studied, which can be useful when investigating how a certain

manipulation might affect drug taking behaviour. In this manner, self-administration

has been a valuable pre-clinical tool in determining the efficacy of purported

treatments for reducing drug consumption. For example, self-administration of a range

of drugs is reduced by vaccines that use the body’s immune system to block drugs

from crossing the blood/brain barrier (Fox et al., 1996; Kantak, 2003; Skolnick, 2015).

Based in part on the results of self-administration studies, a number of these vaccines

have progressed to clinical trials, representing an exciting new potential rehabilitative

tool for reducing drug taking (Heidbreder & Hagan, 2005; Skolnick, 2015).

Overall, the self-administration model allows for direct assessment of drug

taking behaviour, and reduces some of the confounds associated with investigating the

effects of experimenter-administered MDMA on animals (De La Garza et al., 2007;

Fantegrossi, 2007). Furthermore, self-administration of MDMA produces different

neuroadaptations to experimenter administration, and these neuroadaptations are

probably more similar to those produced by regular recreational ecstasy use.

Profile of MDMA self-administration

Just as humans show tremendous variability in their patterns of ecstasy use,

there is considerable variability in the self-administration of MDMA in animals. More

specifically, some individuals are more vulnerable to the reinforcing effects of

MDMA, and the reinforcing efficacy of MDMA appears to increase with repeated

19

exposure. To investigate this variability our laboratory has defined an acquisition

criterion and measured latency to acquisition of MDMA self-administration. To meet

the acquisition criterion a rat must self-administer a total of 90 infusions of MDMA

(1.0 mg/kg/infusion) within 25 self-administration sessions. Our laboratory has shown

that roughly 50% of subjects will acquire MDMA self-administration under these

conditions (Colussi-Mas, Wise, Howard, & Schenk, 2010; Schenk et al., 2012; Schenk

et al., 2003; Schenk et al., 2007). Figure 1.2 presents raw data collected for this thesis

that help to illustrate the variability in acquisition of MDMA self-administration. The

top panel shows the number of MDMA infusions self-administered within the 25

session for a subject (Kererū) that did not meet acquisition criteria. Responding across

the 25 sessions is consistently low, although up to 3 infusions were self-administered

within a session. It should be noted that this rate of self-administration is lower than

that supported by the saline vehicle, which supports roughly 5-7 infusions per session.

The middle panel shows the same data for a subject (Black Stilt) that was slow to

acquire (24 sessions), while the bottom panel shows a subject (Kea) that acquired

relatively quickly (13 sessions). As is typical in these self-administration studies,

initial intake is low in all 3 subjects, but the subjects that did acquire show a sudden

increase in intake. That Kea required less self-administration experience before

increasing intake than Black Stilt shows the underlying variability in the reinforcing

effects of MDMA between these subjects, and this variability is reflected in their

latency to acquisition. The decrease in Kea’s responding in session 7 is typical after

the first exposure to a high dose of MDMA, and can be seen to a lesser extent in the

data from Black Stilt.

20

S e lf-a d m in is tra tio n s e s s io n

Infu

sio

ns

pe

r 2

hr

se

ss

ion

0 5 1 0 1 5 2 0 2 50

5

1 0

1 5

2 0

Subject:  Kererū  

S e lf-a d m in is tra tio n s e s s io n

Infu

sio

ns

pe

r 2

hr

se

ss

ion

0 5 1 0 1 5 2 0 2 50

5

1 0

1 5

2 0

Subject:  Black  Stilt  

S e lf-a d m in is tra tio n s e s s io n

Infu

sio

ns

pe

r 2

hr

se

ss

ion

0 5 1 0 1 5 2 0 2 50

5

1 0

1 5

2 0

Subject:  Kea  

21

Fig. 1.2 Number  of  MDMA  infusions  (1.0  mg/kg/infusion)  self-­‐administered  over  a  25  day  acquisition  period.  Top  panel:  a  subject  that  did  not  acquire.  Middle  panel:  A  subject  that  was  slow  to  acquire  (24  sessions).  Bottom  panel:  A  subject  that  was  relatively  quick  to  acquire  (13  sessions).     Of the subjects that do acquire MDMA self-administration, some self-

administer more reliably than others. In our laboratory once a subject meets the

acquisition criterion the dose of MDMA is halved, and we expect that responding will

compensate accordingly. Often, we will further increase the FR schedule so that more

responses are required to obtain an infusion of drug. Some subjects will not increase

responding as the FR schedule is increased, while in others responding will

compensate for increases in FR (see Chapter 7). Figure 1.3 illustrates these

compensatory increases in responding with data collected for this thesis from a rat that

shows reliable self-administration. The top panel shows that responding compensated

for the decrease in dose (from section A to section B), and for increases in FR values

(sections C and D). The bottom panel shows that total intake becomes consistent over

time, although initially there is some variability, particularly after the first high dose of

MDMA was self-administered.

These patterns of self-administration behaviour have been a focus of our

laboratory for some time. In particular, we find it interesting that some subjects will

increase responding for MDMA after relatively low MDMA intake, while others will

show similar behaviour after relatively high MDMA intake, and others still will not

increase responding for MDMA within our 25 day cut-off period. We have suggested

that this behavioural profile might reflect the pharmacodynamic profile of MDMA.

22

S e lf-a d m in is tra tio n s e s s io n

Le

ve

r p

res

se

s p

er

2 h

r s

es

sio

n

0 5 1 0 1 5 2 0 2 5 3 00

2 0

4 0

6 0

1 2 0

1 4 0

A B C D

S e lf-a d m in is tra tio n s e s s io n

MD

MA

in

tak

e (

mg

/kg

)

0 5 1 0 1 5 2 0 2 5 3 00

5

1 0

1 5

2 0

2 5

Subject:  Kākāpō  

Fig. 1.3 Top  panel:  lever  presses  reinforced  by  MDMA  per  session  across  different  MDMA  doses  and  FR  schedules.  Section  A:  1.0  mg/kg/infusion,  FR  1.  Section  B:  0.5  mg/kg/infusion,  FR  1.  Section  C:  0.5  mg/kg/infusion,  FR  2.  Section  D,  0.5  mg/kg/infusion,  FR  5.  Bottom  panel:  Total  MDMA  intake  over  the  same  self-­‐administration  sessions.

23

Pharmacodynamics of MDMA

MDMA has a diverse pharmacodynamic profile. Battaglia, Brooks,

Kulsakdinun, and De Souza (1988) categorised the 5-HT transporter, 5-HT2 receptors,

α2 adrenergic receptors, and M-1 muscarinic receptors as targets for which MDMA

has high affinity (0-10µM). Moderate affinity (10µM-100µM) targets included the

norepinephrine and dopamine transporters, and 5-HT1 receptors, and low affinity

(>100µM) targets included dopamine-D1 and -D2 receptors and the choline

transporter. A small number of studies have shown that MDMA produces modest

increases in extracellular levels of glutamate (Anneken & Gudelsky, 2012; Nash &

Yamamoto, 1992) and acetylcholine (Acquas et al., 2001; Nair & Gudelsky, 2006a,

2006b), but there is limited evidence for effects on extracellular norepinephrine (Starr,

Page, & Waterhouse, 2012) or GABA (Bankson & Yamamoto, 2004; Yamamoto,

Nash, & Gudelsky, 1995). In contrast, a great deal of research on MDMA has focused

on 5-HT and dopamine mechanisms.

MDMA preferentially releases 5-HT via reverse transport (Gu & Azmitia,

1993; Gudelsky & Nash, 1996; Hekmatpanah & Peroutka, 1990). Although MDMA

has moderate affinity for the norepinephrine and dopamine transporters, MDMA is

more potent at releasing 5-HT (EC50=74.3 nM) than norepinephrine (EC50=136 nM) or

dopamine (EC50=278 nM) (Baumann, Wang, & Rothman, 2007). MDMA also inhibits

the 5-HT transporter (Berger, Gu, & Azmitia, 1992; Rothman & Baumann, 2003),

vesicular monoamine transporter 2 (Bogen, Haug, Myhre, & Fonnum, 2003; Erickson,

Schafer, Bonner, Eiden, & Weihe, 1996; Pifl, Reither, & Hornykiewicz, 2015) and

activity of monoamine oxidase A and B (Leonardi & Azmitia, 1994; Matsumoto et al.,

2014; Scorza et al., 1997). Thus, MDMA enhances extracellular 5-HT levels by

inhibiting the reuptake of 5-HT, directly releasing 5-HT from terminals, inhibiting the

packaging of 5-HT into vesicles, and inhibiting the degradation of 5-HT. Results from

in vivo microdialysis studies reliably show that MDMA preferentially increases

extracellular 5-HT levels (For review see Schenk (2011)). Following acute

administration of MDMA there was an immediate (15 min) and prolonged (2 week)

decrease in tryptophan hydroxylase activity, as measured by a 14CO2-trapping

procedure (Schmidt & Taylor, 1987; Stone, Hanson, & Gibb, 1987; Stone, Johnson,

Hanson, & Gibb, 1988; Stone, Merchant, Hanson, & Gibb, 1987), indicating that

MDMA also inhibits the further production of 5-HT.

24

MDMA produces minor and transient reductions in dopamine transporter

function, as measured in ex vivo synaptosomes, but failed to alter dopamine

transporter binding or tyrosine hydroxylase activity in rats (J. P. Hansen et al., 2002;

Stone, Merchant, et al., 1987). Nonetheless, MDMA administration increases

extracellular dopamine levels, as determined by in vivo microdialysis (for review, see

Schenk (2011)). This increase is more modest than the MDMA-produced increase in

extracellular 5-HT levels. For example, there was a 300% increase in extracellular

dopamine concentrations in the nucleus accumbens following 3 mg/kg MDMA, but an

1800% increase in extracellular 5-HT concentrations (Baumann, Clark, & Rothman,

2008).

A focus on 5-HT

A question remains as to which of these effects of MDMA might be related to

its self-administration. A wealth of data indicate that the reinforcing efficacy of a drug

is directly related to its ability to increase synaptic levels of dopamine. For example,

dopamine agonists reduced self-administration in a manner consistent with a leftward

shift in the dose-response curve (Gardner, 2000; Yokel & Wise, 1978), suggesting

enhanced reinforcement. On the other hand, dopamine antagonists produced

responding consistent with a rightward shift in the dose-response curve (de Wit &

Wise, 1977; Ettenberg, Pettit, Bloom, & Koob, 1982; Gardner, 2000; Yokel & Wise,

1975), suggesting a decrease in reinforcement. Similarly, neurotoxic, 6-OH-DA,

lesions also reduced the reinforcing efficacy of drugs of abuse (Gardner, 2000; Lyness,

Friedle, & Moore, 1979; Roberts, Corcoran, & Fibiger, 1977; Roberts & Koob, 1982).

As is true with other drugs of abuse, the reinforcing efficacy of MDMA, and

thus the self-administration of MDMA, results from dopamine release. As indicated

above, however, MDMA preferentially increases 5-HT, an effect that is incompatible

with self-administration. For example, stimulation of 5-HT release inhibited (Rothman

et al., 2005), while neurotoxic 5,7-DHT lesions enhanced (Bradbury et al., 2014; Loh

& Roberts, 1990) self-administration. Self-administration of amphetamine-type drugs

was inversely related to affinity for the 5-HT transporter (Ritz & Kuhar, 1989), or

potency to stimulate 5-HT release (Wee et al., 2005). With specific reference to

MDMA, the (+) isomer that selectively releases dopamine was more readily self-

administered than the (-) isomer that selectively releases 5-HT (Z. Wang &

Woolverton, 2007). That is, higher levels of 5-HT release are inhibitory to self-

25

administration in general, and to MDMA self-administration in particular. Thus,

MDMA-produced 5-HT release would be expected to inhibit MDMA self-

administration, yet, as outlined above, some rats will eventually self-administer

MDMA reliably. It is possible that some rats are less responsive to these 5-HTergic

effects and so self-administer MDMA more readily.

This hypothesis was recently directly tested in our laboratory. Firstly, the 5-

HTergic response to an initial dose of MDMA was determined by in vivo microdialysis

before MDMA self-administration began. 5-HT release produced by this initial

exposure to MDMA was lower in the rats that did acquire MDMA self-administration

than in those that did not, while dopamine release was similar for both groups.

Secondly, the effect of a neurotoxic 5,7-DHT lesion on acquisition of MDMA self-

administration was determined. The lesion reduced 5-HT tissue levels by up to 67%.

Of interest, 100% of the lesion group acquired MDMA self-administration, compared

to approximately 50% of controls, and the latency to acquisition was greatly reduced in

the lesion group (Bradbury et al., 2014). Thus, lower 5-HT release produced by

MDMA, either endogenous or exogenously produced by a lesion, was associated with

enhanced self-administration. These findings support the hypothesis that MDMA-

produced 5-HT release is inhibitory to the acquisition of MDMA self-administration,

but a question remains as to the mechanism for this inhibitory effect.

It has been suggested that the development of MDMA as an efficacious

reinforcer in the self-administration paradigm is due to neuroadaptations that occur in

response to regular MDMA exposure, and that the same neuroadaptations could

underlie the development of ecstasy SUDs (Schenk, 2011; Schenk & Aronsen, 2015).

Microdialysis studies have shown that the 5-HTergic response to MDMA is attenuated

after repeated exposure (Baumann, Clark, Franken, Rutter, & Rothman, 2008;

Reveron, Maier, & Duvauchelle, 2010; Shankaran & Gudelsky, 1999), an effect that

would be expected to facilitate MDMA self-administration. It has been hypothesised

that this reduced 5-HTergic response to MDMA disinhibits the dopaminergic response,

enhancing the reinforcing efficacy of MDMA and making it comparable to other drugs

of abuse (Schenk, 2011). Furthermore, neuroadaptations in 5-HT receptors, as a result

of MDMA exposure, have been suggested to enhance problematic behaviours, like

impulsivity, that are associated with SUDs (Schenk & Aronsen, 2015).

Thus, repeated exposure to MDMA reduces the 5-HTergic response to MDMA,

enhancing its reinforcing effects and producing behaviours that may contribute to

26

problematic drug taking. If the reinforcing effects of MDMA rely on 5-HTergic

deficits, the variability in acquisition of MDMA self-administration might be due to

increased vulnerability to MDMA-produced 5-HTergic neuroadaptations in some rats.

Because 5-HTergic deficits enhance MDMA self-administration via a disinhibition of

dopamine, there are likely specific 5-HT receptors that modulate the dopaminergic

response to, and thus the self-administration of, MDMA.

There are 14 different 5-HT receptor subtypes, arranged into 7 receptor

families, and spread widely throughout the brain (Hoyer et al., 1994). The 5-HT1A and

5-HT1B receptor subtypes have a role in the regulation of dopamine and the

dopaminergic response to drugs of abuse, and as such changes in the activation of

these receptor subtypes might be expected to alter the reinforcing effects of MDMA.

5-HT1A and 5-HT1B receptors

The 5-HT1A receptor is a seven transmembrane receptor that couples to Gi/Go

to inhibit adenylyl cyclase and produce hyperpolarisation (Hamon et al., 1990; Innis,

Nestler, & Aghajanian, 1988; Schoeffter & Hoyer, 1988). In the brain the 5-HT1A

receptor is located both pre- and post-synaptically. Pre-synaptically, the 5-HT1A

receptor is an autoreceptor on 5-HT neurons in the dorsal and median raphe nuclei,

where activation inhibits 5-HT synthesis, and release of 5-HT in terminal regions

(Hamon et al., 1988; Riad et al., 2000; Yoshimoto & McBride, 1992). 5-HT1A

receptors have also been localised to the hippocampus, amygdala, prefrontal cortex

(PFC), and the ventral tegmental area (VTA) where they act as heteroreceptors on

dopamine, glutamate, and GABA cells (Doherty & Pickel, 2001; Hajós, Gartside,

Varga, & Sharp, 2003; Hume et al., 2001; Maeda et al., 2001; Palchaudhuri & Flügge,

2005; Pompeiano, Palacios, & Mengod, 1992; Puig, Artigas, & Celada, 2005; Puig,

Watakabe, Ushimaru, Yamamori, & Kawaguchi, 2010).

The 5-HT1B receptor is also a seven transmembrane protein that couples to

Gi/Go to inhibit adenylyl cyclase and produce hyperpolarisation (Hartig, Branchek, &

Weinshank, 1992; Hoyer & Middlemiss, 1989; Sari, 2004; Seuwen, Magnaldo, &

Pouysségur, 1988; C. Wang et al., 2013). In the brain the 5-HT1B receptor is located

pre-synaptically on the terminals of 5-HTergic or non-5-HTergic cells, as auto- or

heteroreceptors, respectively (Boulenguez et al., 1996; Offord, Ordway, & Frazer,

1988; Sari et al., 1999; Vergé et al., 1986). 5-HT1B receptor binding was high in globus

pallidus, substantia nigra, nucleus accumbens, frontal cortex, striatum, and

27

hippocampus (Bonaventure, Schotte, Cras, & Leysen, 1997; Lindhe et al., 2011). 5-

HT1B mRNA was also abundant in the hypothalamus, thalamus, and amygdala

(Bonaventure et al., 1998). As well as being present on 5-HTergic neurons, 5-HT1B

receptors have been localised to dopaminergic (Sarhan & Fillion, 1999), GABAergic

(Darrow, Strahlendorf, & Strahlendorf, 1990), and glutamatergic (Raiteri, Maura,

Bonanno, & Pittaluga, 1986) terminals.

Changes in 5-HT1A and 5-HT1B receptor protein levels, mRNA levels, or

binding to G proteins, have been shown in response to general interventions such as

exercise (Chennaoui et al., 2001; Fuss et al., 2013), stress (Iyo et al., 2009; S. Wang,

Zhang, Guo, Teng, & Chen, 2009), and steroid administration (Ambar & Chiavegatto,

2009; Kindlundh, Lindblom, Bergström, & Nyberg, 2003). More importantly, changes

have also been shown after repeated exposure to 5-HTergic ligands. 5-HT1A

autoreceptors were down-regulated by chronic exposure to selective 5-HT reuptake

inhibitors (SSRIs) (Castro, Diaz, del Olmo, & Pazos, 2003; Le Poul et al., 2000),

although no changes in 5-HT1A autoreceptors were detected after repeated exposure to

MDMA (Schenk, Abraham, Aronsen, Colussi-Mas, & Do, 2013). Chronic SSRI

treatment also increased post-synaptic 5-HT1A receptor agonist-stimulated binding of

[35S]GTPγS to G proteins (Castro et al., 2003; Moulin-Sallanon et al., 2009). Similarly,

up-regulation of post-synaptic 5-HT1B receptors has been suggested as a result of

repeated SSRI treatment (Le Poul et al., 2000). These findings suggest that the large

increases in synaptic 5-HT produced by MDMA could also produce changes in these

receptor subtypes.

The role of 5-HT1A and 5-HT1B receptors in dopamine modulation

The most commonly used 5-HT1A receptor agonist, 8-hydroxy-2-

dipropylaminotetralin (8-OH-DPAT), has high affinity for 5-HT1A receptors (Peroutka,

1986). Low doses of 8-OH-DPAT preferentially activated 5-HT1A autoreceptors, while

higher doses also activated heteroreceptors (Alex & Pehek, 2007; Hjorth &

Magnusson, 1988). Low doses of 8-OH-DPAT simulated dopamine cell activity in the

VTA (Gronier, 2008) and increased extracellular dopamine concentrations in the PFC

(Arborelius, Nomikos, Hacksell, & Svensson, 1993) and VTA (Chen & Reith, 1995).

Thus, activation of 5-HT1A autoreceptors enhances dopamine cell activity and

extracellular dopamine concentrations.

Higher doses of 8-OH-DPAT inhibited dopamine cell firing in the VTA

(Arborelius, Chergui, et al., 1993) and decreased extracellular dopamine levels in the

28

nucleus accumbens (NAc) (Ichikawa & Meltzer, 2000) and striatum (Rasmusson,

Goldstein, Deutch, Bunney, & Roth, 1994), as measured by in vivo microdialysis. The

relatively new 5-HT1A receptor agonists, F13640, and F15599, both have >1000 fold

selectivity for the 5-HT1A receptor (Colpaert et al., 2002; Newman-Tancredi et al.,

2009), while the slightly older BAY × 3702 has approximately 30 fold selectivity for

the 5-HT1A receptor (De Vry et al., 1998). Each of these agonists, when administered

locally in the PFC, dose-dependently increased extracellular dopamine levels in the

PFC (Díaz-Mataix, Artigas, & Celada, 2006; Díaz-Mataix et al., 2005; Lladó-Pelfort,

Assié, Newman-Tancredi, Artigas, & Celada, 2012; Lladó-­‐Pelfort, Assié, Newman-­‐

Tancredi, Artigas, & Celada, 2010). It was suggested that this effect was due to

inhibition of PFC GABA and glutamate cells, since endogenous 5-HT release inhibited

electrophysiological recordings from PFC glutamate and GABA cells, and this effect

was attenuated by a 5-HT1A receptor antagonist (Hajós et al., 2003; Puig et al., 2005;

Puig et al., 2010; Sakaue et al., 2000). Thus, the effect of 5-HT1A receptor activation

on dopamine release is region specific – activation of autoreceptors, or heteroreceptors

in the PFC, increased extracellular dopamine concentrations, while global activation of

heteroreceptors decreased extracellular dopamine concentrations in the NAc and

striatum.

There is evidence that activation of 5-HT1A receptors is inhibitory to the

dopaminergic response to drugs of abuse. The 5-HT1A receptor agonist, 8-OH-DPAT,

inhibited amphetamine-induced dopamine release in the PFC (Kuroki, Ichikawa, Dai,

& Meltzer, 1996), striatum and NAc (Ichikawa, Kuroki, Kitchen, & Meltzer, 1995) as

determined by microdialysis. 5-HT1A receptor agonists generally inhibited the

hyperactive response to amphetamine, methamphetamine, and MDMA (Müller, Carey,

Huston, & Silva, 2007), a response that has been associated with enhanced dopamine

neurotransmission (Wise & Bozarth, 1987). Furthermore, the expression and

development of cocaine or amphetamine sensitisation in mice was inhibited by 5-HT1A

receptor agonist administration (Ago et al., 2006; Przegaliñski, Siwanowicz, Baran, &

Filip, 2000). Thus, increased activation of 5-HT1A receptors during MDMA self-

administration might be expected to inhibit the dopaminergic response to MDMA.

In vitro studies showed that activation of 5-HT1B receptors inhibited the release

of dopamine (Sarhan & Fillion, 1999), GABA (Johnson, Mercuri, & North, 1992; Yan

& Yan, 2001b), and glutamate (Muramatsu, Lapiz, Tanaka, & Grenhoff, 1998), but

these studies do not consider interactions between neurotransmitter systems. One of

29

the most widely used 5-HT1B receptor agonists, RU 24969 (5-Methoxy-3-(1,2,5,6-

tetrahydro-4-pyridinyl)-1H-indole), has high affinity for 5-HT1B receptors (Ki = 0.38

nM), but also displays appreciable affinity for the 5-HT1A receptor (Ki = 2.5 nM)

(Peroutka, 1986; Wolf & Kuhn, 1991). Systemic administration of RU 24969

decreased extracellular GABA concentrations in the VTA in vivo, but had no effect on

extracellular dopamine concentrations (Parsons, Koob, & Weiss, 1999). However,

local administration of the 5-HT1B receptor agonist, CP 93129, which has 150 fold

selectivity for 5-HT1B over other 5-HT receptors (Macor et al., 1990), increased

extracellular dopamine concentrations in the PFC (Iyer & Bradberry, 1996), striatum

(Galloway, Suchowski, Keegan, & Hjorth, 1993), and NAc (Hållbus, Magnusson, &

Magnusson, 1997; Yan & Yan, 2001a). Similarly, administration of CP 93129 in the

VTA increased extracellular dopamine levels in the NAc (O'Dell & Parsons, 2004;

Yan & Yan, 2001a; Yan, Zheng, & Yan, 2004) and decreased extracellular GABA

concentrations in the VTA (O'Dell & Parsons, 2004; Yan et al., 2004), without altering

extracellular glutamate concentrations in the VTA (O'Dell & Parsons, 2004). Together

these findings suggest that activation of 5-HT1B receptors enhances dopamine release,

possibly via an inhibition of GABA neurotransmission.

There is evidence that activation of 5-HT1B receptors enhances the

dopaminergic response to drugs of abuse. Cocaine produced significantly greater

increases in extracellular dopamine, and significantly greater reductions in

extracellular GABA, in the NAc after systemic administration of the 5-HT1B/1A

receptor agonist, RU 24969 (Parsons et al., 1999). A similar response to cocaine was

found after infusion of the 5-HT1B receptor agonist, CP 93129, in the VTA (O'Dell &

Parsons, 2004). Systemic administration of the 5-HT1B receptor agonist, CP 94253,

which has approximately 45 fold selectivity for 5-HT1B over other 5-HT receptors

(Koe, Nielsen, Macor, & Heym, 1992), significantly prolonged the increase in

extracellular dopamine in the NAc produced by systemic administration of ethanol

(Yan, Zheng, Feng, & Yan, 2005). 5-HT1B receptor agonists produced a leftward shift

in the cocaine self-administration dose response curve, increased the break points

achieved in cocaine progressive ratio tasks (Parsons, Weiss, & Koob, 1998;

Pentkowski, Acosta, Browning, Hamilton, & Neisewander, 2009; Przegaliñski, Gołda,

Frankowska, Zaniewska, & Filip, 2007), and produced a leftward shift in the self-

administration dose response curve for the dopamine uptake inhibitor, GBR 12909

(Parsons, Weiss, & Koob, 1996). Therefore, activation of 5-HT1B receptors during

30

MDMA self-administration might be expected to enhance the dopaminergic response

to MDMA.

Thus, 5-HT1A and 5-HT1B receptors might be expected to impact the initial

reinforcing effects of MDMA, via dopaminergic modulation. Specifically, activation

of 5-HT1A receptors would be expected to decrease the dopaminergic response to

MDMA, while activation of 5-HT1B receptors would be expected to enhance the

dopaminergic response to MDMA. Furthermore, alterations in the activation of these

receptors might explain the enhancement in the reinforcing efficacy of MDMA after

repeated exposure. As outlined below, individual variability in these receptor

populations, or MDMA-produced changes in these receptor populations, might also

impact the reinforcing effects of MDMA and explain behavioural deficits seen in

regular ecstasy users.

5-HT1A and 5-HT1B receptors and impulsivity

Impulsivity has been broadly defined as action without foresight, referring to

behaviours that are poorly thought out, prematurely executed, or risky (Winstanley,

Eagle, & Robbins, 2006). In drug users impulsivity is a risk factor for initiating drug

taking, escalating drug use, and for developing SUDs (De Wit, 2009; Perry & Carroll,

2008). For example, impulsive traits in youth and young-adulthood positively

predicted future drug use, an earlier onset of drug taking, and the likelihood of

developing an SUD (De Wit, 2009; Kirisci, Tarter, Mezzich, & Vanyukov, 2007;

Sher, Bartholow, & Wood, 2000; Tarter, Kirisci, Feske, & Vanyukov, 2007).

A role of impulsivity in different aspects of drug self-administration in animals

has been determined. Some studies have looked at the acquisition and maintenance of

self-administration, based on the idea that highly impulsive subjects, as is the case with

humans, might be more prone to take drugs (Perry & Carroll, 2008). Typically,

impulsivity is measured by a model of behavioural inhibition, such as the 5 choice

serial reaction time task (5CSRTT), or a model of choice preference for a delayed

reward, such as the delay discounting paradigm. These measures show good validity as

they are variants of those used to assess aspects of impulsive behaviour in humans

(Evenden, 1999b; Robbins, 2002). Delay discounting and reaction time tasks can be

used to determine impulsivity scores across a group of animal subjects, which can then

be divided into ‘low impulsivity’ (LI) groups and ‘high impulsivity’ (HI) groups. HI

subjects are usually defined as those in the upper quartile of impulsivity scores, with

LI subjects being those with impulsivity scores in the bottom quartile. These two

31

groups can then be compared to determine the relationship between impulsivity and

drug self-administration.

When impulsivity was determined using a delay discounting task HI rats

consumed more ethanol (Poulos, Le, & Parker, 1995), or cocaine (Koffarnus &

Woods, 2013; Perry, Larson, German, Madden, & Carroll, 2005; Perry, Nelson, &

Carroll, 2008), and cocaine self-administration was acquired more quickly and in a

higher percentage of HI rats (Perry et al., 2005; Zlebnik & Carroll, 2015). Similarly,

HI rats, as measured by 5CSRTT performance, acquired nicotine self-administration

more readily (Diergaarde et al., 2008), and a strain of mice with high impulsivity

showed enhanced ethanol self-administration (Loos, Staal, Smit, De Vries, & Spijker,

2013). Following acquisition, HI rats, as determined by the 5CSRTT, self-administered

more cocaine per hour than LI rats, and exhibited an upward shift in the cocaine dose

response curve (Dalley et al., 2007). Furthermore, impulsivity as determined by the

5CSRTT predicted the magnitude of the drug-seeking response for MDMA in the

reinstatement paradigm (Bird & Schenk, 2013). Thus, higher levels of impulsivity

would be expected to facilitate self-administration.

Systemic administration of the 5-HT1A receptor agonist, 8-OH-DPAT,

increased premature responding on the 5CSRTT (Carli & Samanin, 2000) while the 5-

HT1A receptor antagonist, WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-

piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide), which has >100-fold

selectivity for the 5-HT1A receptor over other receptors (A. Fletcher et al., 1995),

supressed impulsive action in a 3CSRTT (Ohmura et al., 2013). The effects of 5-HT1A

ligands on impulsivity appear to be due to autoreceptor activation, because neither

local administration of 8-OH-DPAT in the PFC, nor systemic administration of the 5-

HT1A post-synaptic preferring receptor agonist F15599, affected premature responding

on the 5CSRTT (Carli, Baviera, Invernizzi, & Balducci, 2006; Lladó-­‐Pelfort et al.,

2010; Winstanley et al., 2003).

In humans, 5-HT1B receptor gene polymorphisms are associated with impulsive

aggression (Zouk et al., 2007). Mice that lack the 5-HT1B receptor gene from birth

show increased impulsivity in a behavioural model of response inhibition (Nautiyal et

al., 2015; Pattij et al., 2003). Interestingly, knockdown of 5-HT1B autoreceptors did not

affect impulsivity, suggesting the effect of 5-HT1B receptor activation on impulsivity is

due to heteroreceptor action (Nautiyal et al., 2015). Studies of the effects of 5-HT1B

ligands on impulsivity have been limited due to the fact that agonists have a range of

32

behavioural effects that disrupt operant responding (Evenden, 1999a; van den Bergh,

Bloemarts, Groenink, Olivier, & Oosting, 2006). However, the limited available data

suggest that activation of 5-HT1B receptors reduces impulsive behaviour (Evenden,

1999a). Therefore, activation of 5-HT1B receptors during MDMA self-administration

would be expected to reduce impulsive behaviour, and thus inhibit self-administration.

5-HT1A and 5-HT1B receptors and learning

Before reliable self-administration behaviour can be demonstrated, the subject

must learn the association between performance of the operant response and the

infusion of drug. Enhanced or inhibited ability to learn this association would clearly

also enhance or inhibit the acquisition of self-administration. There is also another

learned association that has been shown to be incredibly important in the self-

administration paradigm - the Pavlovian association between the drug effect and the

contextual stimuli (e.g. the light). These unconditioned contextual stimuli develop

conditioned reinforcement properties over repeated pairings with a drug (Ahrens,

Singer, Fitzpatrick, Morrow, & Robinson, 2016; W. M. Davis & Smith, 1976; P. J.

Fletcher & Korth, 1999b) and these conditioned reinforcers are a powerful driver of

self-administration behaviour. For example, one experiment assessed the acquisition of

nicotine self-administration in two groups of rats – one in which the nicotine infusion

was paired with the illumination of a light, and another in which the infusion was

paired with no specific cues. Rats in the nicotine + cue group took less time to show a

preference for the active self-administration lever and consumed significantly more

nicotine than the nicotine only group, suggesting the Pavlovian association between

drug effect and contextual cues facilitated acquisition of self-administration (Caggiula

et al., 2002). Therefore, enhanced or inhibited learning of either operant or Pavlovian

associations would be expected to enhance or inhibit self-administration, respectively.

The strengthening of stimulus/reward associations is markedly impacted by

pharmacological manipulation of 5-HT1A receptors. Systemic 5-HT1A receptor agonist

administration impaired performance on an appetitive Pavlovian conditioned

responding task (Blair, Bonardi, & Hall, 2004), increased errors in a repeated

acquisition of response sequence task (Winsauer, Rodriguez, Cha, & Moerschbaecher,

1999) and delayed acquisition of operant responding maintained by a food reinforcer

(Frick, Bernardez-Vidal, Hocht, Zanutto, & Rapanelli, 2015). Furthermore, the 5-HT1A

receptor agonist, 8-OH-DPAT, administered after an initial training session, impaired

further operant responding for food (Meneses, 2007). The lack of comprehensive dose-

33

response functions in these studies limits the degree to which the relative roles of 5-

HT1A auto- and heteroreceptors can be disentangled. Importantly, the above results

were noted over a range of 8-OH-DPAT doses that would be expected to activate pre-

and post-synaptic 5-HT1A receptors (up to 1.0 mg/kg). When low doses of 8-OH-

DPAT were used, operant learning was enhanced (Meneses & Hong, 1994b), and this

effect was reversed by the tryptophan hydroxylase inhibitor, pCPA (Meneses & Hong,

1994a). Together, these results suggest that activation of 5-HT1A autoreceptors

enhances, while activation of 5-HT1A heteroreceptors inhibits, learning of

stimulus/reward associations.

The non-selective 5-HT receptor agonist, mCPP, inhibited operant

stimulus/response learning, and this effect was reversed by the non-selective 5-HT1B

receptor antagonist, propranolol (Meneses & Hong, 1997). Moreover, the 5-HT1B

receptor agonist, CGS 12066 impaired (Meneses, 2007), while the 5-HT1B/1D receptor

antagonist, GR 127935, improved (Meneses, Terrón, & Hong, 1997) performance on

the same task. Similarly, the 5-HT reuptake facilitator, tianeptine, enhanced operant

stimulus/response learning, and this effect was reversed by the 5-HT1B receptor inverse

agonist, SB 224289 (Meneses, 2002). These findings suggest that activation of 5-HT1B

receptors inhibits the consolidation of operant learning, and so activation of 5-HT1B

receptors during MDMA self-administration might be expected to inhibit the

development of self-administration.

5-HT1A and 5-HT1B receptors and anxiety

Anxiety disorders are frequently comorbid with SUDs (Ipser, Wilson,

Akindipe, Sager, & Stein, 2015; Merikangas et al., 1998). It has been suggested that

anxiety may underlie the initiation of drug taking, in order to alleviate a negative

emotional state, and negatively reinforce the continuation of drug use to mitigate

withdrawal symptoms (Altman et al., 1996; Belin, Belin-­‐Rauscent, Everitt, & Dalley,

2015; Lejuez et al., 2008). In animal models, anxiety is often operationalised in

rodents as an aversion to open or brightly lit spaces (Belin et al., 2015). A popular

method for measuring anxiety is the elevated plus maze (EPM), in which a preference

for the closed (protected) arms of the maze over the open arms is regarded as an

‘anxious’ response (Pellow, Chopin, File, & Briley, 1985). An alternate measure of

rodent anxiety is self-grooming behaviour in response to an environmental change

(Homberg et al., 2002). Rats in the upper quartile for time spent grooming in a novel

environment reached higher break points in progressive ratio cocaine self-

34

administration than the lower quartile group (Homberg et al., 2002). This effect was

not replicated when high anxiety was determined by performance on the EPM,

however in this case high and low anxiety were determined using a median split, thus

possibly masking an effect of anxiety (Bush & Vaccarino, 2007). Higher anxiety on

the EPM was associated with escalation of cocaine self-administration (Dilleen et al.,

2012), and propensity to self-administer alcohol (Spanagel et al., 1995). Thus, higher

levels of anxiety would be expected to facilitate self-administration.

Time spent in the open arms of the EPM was increased by systemic

administration of low doses of the 5-HT1A receptor agonist, 8-OH-DPAT (Kwieciński

& Nowak, 2009; Lalonde & Strazielle, 2010), an effect reversed by the 5-HT1A

receptor antagonist, WAY 100635 (Collinson & Dawson, 1997), suggesting 5-HT1A

autoreceptor activation had an anxiolytic effect. Higher doses of systemically

administered 8-OH-DPAT had an anxiogenic effect in the same task in mice (Miheau

& Van Marrewijk, 1999).When injected into the dorsal or median raphe, 8-OH-DPAT

increased time spent in the open arms of the EPM (De Almeida, Giovenardi, Charchat,

& Lucion, 1998; File & Gonzalez, 1996; File, Gonzalez, & Andrews, 1996), while

injections into the hippocampus (Cheeta, Kenny, & File, 2000a; File et al., 1996; File,

Kenny, & Cheeta, 2000), PFC (Solati, Salari, & Bakhtiari, 2011), or septum (Cheeta,

Kenny, & File, 2000b; De Almeida et al., 1998) increased anxiety-like behaviour in

the EPM. Thus, activation of 5-HT1A autoreceptors had anxiolytic effects in the EPM,

while activation of post-synaptic 5-HT1A receptors was anxiogenic.

Early investigations of the role of 5-HT1B receptors in anxiety states found that

non-selective 5-HT1B receptor agonists decreased time spent in the open arms of an

EPM (Benjamin, Lal, & Meyerson, 1990; Critchley & Handley, 1987; Pellow,

Johnston, & File, 1987), suggesting that activation of 5-HT1B receptors was

anxiogenic. The role of 5-HT1B receptors in modulating anxiety was more recently

confirmed; entries into the open arms of the EPM were dose-dependently reduced by

the 5-HT1B receptor agonist CP 94253, and this effect was reversed by the 5-HT1B/1D

receptor antagonist, GR 127935 (Lin & Parsons, 2002). The relative contribution of 5-

HT1B auto- and heteroreceptors to this effect is not clear. 5-HT1B heteroreceptors on

GABAergic amygdala neurons have been suggested as a possible neuronal mechanism

(Lin & Parsons, 2002; Sari, 2004) because 5-HT1B manipulations of these projections

altered behaviour in the EPM (Audi, De Oliveira, & Graeff, 1991). Furthermore,

activation of 5-HT1B receptors in the PFC produced anxiogenic effects in the EPM

35

(Solati et al., 2011) however, a role of 5-HT1B autoreceptors cannot be ruled out (Sari,

2004).

Summary

MDMA is widely used recreationally in the form of the street drug, ecstasy.

Although the majority of users consume ecstasy intermittently, there is concern that

MDMA produces a range of deficits in regular ecstasy users. Among these deficits,

ecstasy users show increased anxiety and impulsivity, and impaired learning and

memory. Problematically, these behavioural changes might be expected to facilitate

further ecstasy taking.

MDMA is unique among drugs of abuse in that it primarily acts as a 5-HT

releasing agent. 5-HT release has been hypothesised to inhibit the self-administration

of drugs in general, and of MDMA in particular. Nonetheless, MDMA self-

administration is acquired in roughly 50% of animal subjects. It is possible that

MDMA-produced 5-HT release inhibits the reinforcing efficacy of MDMA via

activation of specific 5-HT receptors, but there is likely variability in the 5-HTergic

response to MDMA between individuals. Furthermore, it is possible that

neuroadaptations in 5-HT receptors underlie both the facilitated reinforcement

produced by MDMA after repeated exposure, and the cognitive and behavioural

deficits seen after regular use.

The 5-HT1A and 5-HT1B receptors are good candidates for these effects of

MDMA. Both receptors modulate the reinforcing effects of other drugs of abuse by

regulating dopamine release. Furthermore, these receptors mediate a number of

behaviours associated with self-administration that are impacted by regular ecstasy

use, and receptor up- or down-regulation has been documented in response to a

number of different interventions.

This thesis will explore two ways in which alterations in 5-HT1A and/or 5-HT1B

receptors could influence MDMA self-administration. Firstly, underlying differences

in 5-HT1A and/or 5-HT1B receptors could predispose some subjects to self-administer

more readily. This may explain the variability in acquisition of MDMA self-

administration. If so, it is hypothesised that manipulations that alter 5-HT1A and/or 5-

HT1B receptors will reduce the variability in the acquisition profile for MDMA self-

administration.

36

Secondly, MDMA exposure during self-administration might produce changes

in these receptor populations that might facilitate further drug taking and produce

behavioural deficits. If so, it is hypothesised that these changes will be evident after

substantial MDMA self-administration. Theoretically, if these changes are not the

result of neurotoxicity, they could be partially reversed by repeated administration of

selective agonists or antagonists.

37

General Methods

Subjects

Male Sprague-Dawley rats were bred in the Victoria University of Wellington

vivarium. They were housed in groups of 4 in a temperature- (19-21ºC) and humidity-

(55%) controlled environment until they reached weights of 300-350g, after which

they were housed individually. The housing colony was maintained on a 12 h

light/dark cycle (lights on at 7.00 am) and all tests were conducted during the light

portion of the cycle. Food and water were freely available except during testing.

Drugs

RU 24969 hemisuccinate, WAY 100635 maleate, lithium chloride, ±8-OH-

DPAT hydrobromide, (Tocris, New Zealand), and d-amphetamine sulfate (BDG, New

Zealand) were dissolved in sterilised saline. GR 127935 hydrochloride (Tocris, New

Zealand) was dissolved in distilled water. All injections were a volume of 1.0 ml/kg.

±MDMA hydrochloride (BDG, New Zealand) for self-administration was dissolved in

sterilised saline containing 3IU heparin per ml.

All doses refer to salt weights.

Apparatus and procedures

Water consumption

Water consumption was measured in the home cage. Water bottles were

removed for 24 hours. Drug administration occurred before water bottles were

reintroduced, at times specified in each study. Consumption was measured for a 30

minute period. Fluid consumption was determined by weighing water bottles before

and after the test.

Locomotor Activity

Locomotor activity testing was conducted in clear Plexiglas chambers (Med

Associates Inc., USA; model ENV-515) measuring 42×42×30 cm, set in sound-

attenuating boxes. Forward locomotion was measured with two sets of 16 infrared

beams and sensors spaced evenly along the sides of the chambers producing squares

measuring 25mm × 25mm. The interruption of three adjacent beams (the approximate

size of the body of a rat) was recorded as one activity count. A white noise generator

was used during experiments to mask any outside noise, and chambers were washed

with Virkon ‘S’ disinfectant (Southern Veterinary Supplies, NZ) after testing to control

for olfactory confounds. Experiments were run in a dark room, except for a red light

38

that was used to illuminate the room during drug administrations. Locomotor activity

counts were recorded in 5 minute intervals.

Surgery

For rats that underwent self-administration testing, a silastic catheter was

implanted into the right jugular vein under deep anesthesia produced by i.p. injection

of ketamine (90 mg/kg) and xylazine (9 mg/kg). Areas surrounding skin that was to be

cut were shaved and washed with ethanol and iodine, and eye lubricant (Refresh lacri-

lube) was administered to avoid drying. The catheter was secured in place using

surgical string and a small amount of adhesive (Bostick superglue). The distal end of

the catheter was passed subcutaneously to an exposed part of the skull, attached to a 3

cm piece of 22 gauge stainless steel tubing (BD needles), fixed in place with screws

and a small amount of adhesive, and embedded in dental acrylic (Ostron 100). The

silastic tubing was coated with silicone (Selleys wet area silicone) to protect from the

corrosive nature of the adhesive. Following surgery an analgesic (Carporfen ®, 5.0

mg/kg, s.c.) and electrolyte replacement (Hartman’s solution, 12 ml, s.c.) were

administered. Carprofen was also administered on each of two days following the

surgery. Testing began once pre-surgery weight had been attained, generally within 4-

6 days.

Self-administration

Every day, before self-administration testing, rats were weighed and

administered penicillin dissolved in heparinised saline (0.2 ml, i.v.) to help maintain

general health and catheter patency.

Self-administration was conducted in operant chambers (Med Associates ENV-

001) equipped with two levers. Depression of the active lever resulted in a 12 second

activation of a syringe pump (Razell, Model A, 1 RPM) resulting in a 0.1 ml

intravenous infusion, and the simultaneous illumination of the house light located

above the active lever. Depressions of the inactive lever were recorded, but had no

programmed consequence. Each self-administration session began with an

experimenter-delivered infusion to fill the volume of the catheter. These infusions are

not recorded and do not contribute to calculations of total self-administration intake.

39

Chapter 3: Development of behavioural assays

These first studies were designed to develop behavioural assays for 5-HT1A and

5-HT1B receptor activation, so that further testing of the effects of drug exposure on the

function of these receptor subtypes could be conducted. One assay that seemed

promising was latent inhibition: the impairment of learning that a stimulus predicts an

important event when that stimulus has previously been presented with no

consequence (Cassaday, Hodges, & Gray, 1993).

When a neutral stimulus, for example a tone, is paired with a negative

consequence, for example a footshock, that stimulus will develop conditioned-stimulus

properties and produce freezing behaviour in rodents. A group of rats that had never

been exposed to the tone (control group) would learn this association relatively

quickly. However, if the tone has previously been presented to another group of rats

without consequence (pre-exposure group), learning that the tone now predicts a

footshock will take longer in this group. Therefore, after a small number of pairings,

the tone will produce less freezing behaviour in the pre-exposure group, because the

association between the tone and the footshock is less well learned. Latent inhibition

can be operationalised as this behavioural difference (reduced freezing behaviour)

between groups. Latent inhibition is a robust effect, found across a range of stimulus-

consequence combinations in a wide range of species (Fernández, Giurfa, Devaud, &

Farina, 2012; Ferrari & Chivers, 2011; Lubow, 1989).

A number of studies have implicated 5-HT in latent inhibition. Electrolytic, or

neurotoxic 5,7 DHT, lesions of the median raphe or NAc blocked the latent inhibition

effect (Loskutova, 2001; Loskutova, Luk'yanenko, & Il'yuchenok, 1990; Solomon,

Nichols, Kiernan, Kamer, & Kaplan, 1980). Rats in the pre-exposure group showed

greater 5-HT metabolism in the striatum and amygdala than rats in the control group,

suggesting the latent inhibition effect is associated with increased 5-HTergic activity

(Molodtsova, 2003). Additionally, rats with a genetic deletion of the 5-HT transporter

showed reduced latent inhibition compared to wildtype counterparts (Nonkes et al.,

2012).

The role of 5-HT in latent inhibition is reinforced by studies employing

selective pharmacological ligands. The 5-HT1B/1A receptor agonist, RU 24969 (0.5

mg/kg), administered before each pre-exposure, inhibited the development of latent

inhibition, while the more selective 5-HT1A receptor agonist, 8-OH-DPAT, had no

significant effect (Cassaday et al., 1993), demonstrating a role of 5-HT1B receptors in

40

latent inhibition. On the other hand, the 5-HT1A receptor antagonist, WAY 100635 (0.5

mg/kg), facilitated latent inhibition in the same task (Killcross, Stanhope, Dourish, &

Piras, 1997), suggesting 5-HT1A receptors also impact the expression of latent

inhibition. Thus, RU 24969 inhibited the development of latent inhibition, but the

relative roles of 5-HT1A and 5-HT1B receptor activation in this effect, and the range of

doses over which this effect is produced, have not been determined.

The majority of studies have employed a footshock when investigating latent

inhibition. Given the well-established role of 5-HT, and particularly 5-HT1A receptors,

in the processing of pain (Avila-Rojas et al., 2015; Colpaert, 2006; Colpaert et al.,

2002; Panczyk et al., 2015), I wanted an alternative paradigm to test for latent

inhibition. Latent inhibition can be readily demonstrated using the conditioned taste

aversion paradigm. Conditioned taste aversion refers to the phenomenon whereby an

unfamiliar taste (e.g. a new blend of coffee), paired with a negative internal state (e.g.

feeling sick after drinking), results in future avoidance of that taste (the new coffee).

This association is rapidly learned if the taste stimulus is novel, but this learning is

hampered if the taste stimulus has previously not predicted the illness (e.g. your usual

coffee blend). Thus, prior exposure to the neutral stimulus, without negative

consequence, inhibits the learning of an association, and so latent inhibition can be

demonstrated through an attenuated conditioned taste aversion. In rats this is typically

achieved by pairing flavoured water with a drug (e.g. lithium chloride) that produces

“internal malaise” (Lubow, 1989, p. 5). One pairing of the flavoured water with the

drug is sufficient to ensure the animal avoids the flavoured water in the future, but this

effect is attenuated if, previously, the flavoured water has been consumed without

consequence (Ellenbroek, Knobbout, & Cools, 1997; Mora et al., 1999). This

paradigm has been successfully used to investigate the effects of antipsychotic-type

drugs on latent inhibition (Geyer & Ellenbroek, 2003; Moser, Hitchcock, Lister, &

Moran, 2000).

As a first attempt to assess the roles of 5-HT1A and 5-HT1B receptors in latent

inhibition, the effect of the 5-HT1B/1A receptor agonist, RU 24969, on latent inhibition

in a conditioned taste aversion paradigm was tested.

41

Method

Procedure

Firstly, I aimed to establish the conditioned taste aversion effect, based on the

methods of Ellenbroek et al. (1997). All testing was conducted in the home cages.

Water bottles were removed from the home cages and made available for 30 minutes

per day. Rats (see general methods, n=8 per group) were randomly assigned to have

either water (water pre-exposure group) or a 5% sucrose solution (sucrose pre-

exposure group) available for drinking. Water bottles were weighed before and after

each 30 minute drinking period to measure consumption. Once total consumption

during this pre-exposure phase reached 40ml (approximately 3 days; Ellenbroek et al.

(1997)), rats in both groups received the 5% sucrose solution for 30 minutes.

Immediately after this 30 minute drinking period, lithium chloride (75 mg/kg, i.p.;

Ellenbroek et al. (1997)) was administered. The next day both water and the sucrose

solution were made available for 30 minutes. Taste aversion was measured as the

proportion of sucrose consumed on this test day (amount of sucrose solution consumed

divided by total fluid consumption), with lower proportions of sucrose consumption

indicative of greater taste aversion. Thus, latent inhibition was indicated by a lower

taste aversion (i.e. greater proportion of sucrose consumption) in the sucrose pre-

exposure group.

Other groups were tested to determine the effect of RU 24969 pretreatment on

this latent inhibition effect. The same protocol were used, but 15 minutes prior to water

bottles being available during the pre-exposure phase, rats were injected with RU

24969 (0, 0.03, 0.3, 3.0 mg/kg, s.c.). This range of RU 24969 doses has been shown to

be behaviourally effective in different paradigms (Kennett, Dourish, & Curzon, 1987;

Tricklebank, Middlemiss, & Neill, 1986). The 15 minute RU 24969 pretreatment time

is common (Acosta, Boynton, Kirschner, & Neisewander, 2005; P. J. Fletcher &

Korth, 1999b), because maximal effects have been shown between 15 minutes and 4

hours after administration (Tricklebank et al., 1986).

Statistical analyses

Fluid consumption was compared as a function of pre-exposure using one-way

analysis of variance (ANOVA). A 3 (RU 24969 dose) × 3 (Session) mixed model

ANOVA, with session as the within subjects factor, was used to analyse the effect of

RU 24969 on fluid consumption as a function of pre-exposure session. Where

appropriate, post-hoc analyses were conducted using Tukey’s HSD method.

42

Results

Experiment 1: Pilot study on the Conditioned Taste Aversion effect

Rats in both pre-exposure groups (n=8 per group) met the fluid consumption

criterion in 3 daily pre-exposure sessions. There was no difference in total fluid

consumption between the sucrose and water groups (F(1,14)=2.10, p=0.17). Figure 3.1

shows the water pre-exposure group demonstrated conditioned taste aversion, as

indicated by the low proportion of sucrose consumed on the test day. The sucrose pre-

exposure group showed significantly greater sucrose consumption than the water pre-

exposure group (F(1,14)=4.53, p=0.05, ɳp2= 0.25). Figure 3.1 shows that the sucrose

pre-exposure group consumed similar amounts of water and sucrose on the test day,

suggesting the internal malaise produced by lithium chloride was not associated with

the sucrose solution, thus illustrating latent inhibition.

Sucros

eWate

r0.0

0.2

0.4

0.6

0.8

Pre-exposure

Prop

ortio

n su

cros

e

*

Fig. 3.1 Conditioned  taste  aversion  to  sucrose  after  pairing  with  lithium  chloride  in  rats  either  pre-­‐exposed  to  sucrose  or  water.  The  lack  of  preference  for  water  over  sucrose  in  the  sucrose  pre-­‐exposure  group  is  indicative  of  latent  inhibition.  n  =  8  per  group,  error  bars  represent  SEM.  *p=0.05   Experiment 2: The effect of RU 24969 on Conditioned Taste Aversion

Only 11 subjects were available at the beginning of this experiment, so they

were divided into groups that received different doses of RU 24969 (0.0, 0.03, 0.3, 3.0

mg/kg, s.c.) and different pre-exposures (water, sucrose; n=2-3 per group) with the

intention of adding more subjects to each group as they became available. However,

43

the initial groups treated with RU 24969 appeared to consume less fluid in the daily 30

minute sessions. Figure 3.2 shows the fluid consumption over the first 3 sessions

(collapsed across pre-exposure group) as a function of RU 24969 dose. It is clear that

higher RU 24969 dose groups initially consumed less fluid than the lower dose groups.

ANOVA confirmed a significant effect of RU 24969 dose (F(3,54)=25.8, p<0.001,

ɳp2=0.59), and a post-hoc Tukey test showed that the 3.0 and 0.3 mg/kg RU 24969

dose groups both consumed less fluid than the 0.0 and 0.03 mg/kg groups across the

first 3 sessions. This decrease in fluid consumption provided a confound that would

compromise interpretation of a conditioned taste aversion experiment. Therefore, no

further testing was conducted.

P re -e x p o s u r e s e s s io n

Da

ily

flu

id c

on

su

mp

tio

n

(ml)

1 2 30

5

10

15

20

0 .0

0 .0 3

0 .3

3 .0

R U 2 4 9 6 9 (m g /k g )

Fig 3.2 The  effect  of  RU  24969  dose  on  the  amount  of  fluid  consumed  by  fluid-­‐deprived  rats  during  the  first  3  drinking  sessions.  RU  24969  dose-­‐dependently  decreased  fluid  consumption,  with  the  0.3  and  3.0  mg/kg  groups  consuming  significantly  less  fluid  over  the  3  sessions.  n=4-­‐6  per  group,  error  bars  represent  SEM.  Discussion

The latent inhibition effect was successfully produced using the conditioned

taste aversion paradigm. However, the impact of RU 24969 on this effect could not be

assessed because higher doses of RU 24969 reduced fluid consumption. These results

showed that the conditioned taste aversion paradigm was a confounded assay for

measuring behavioural responses to RU 24969 under these conditions.

Serendipitously, the results also suggested a more straightforward measure of

5-HT1B activation, that of reduced drinking, or adipsia. This response to RU 24969 had

44

been alluded to in the literature. For example, RU 24969 non-selectively reduced

intake of both water and sweetened ethanol (Silvestre, Palacios, Fernandez, & O'Neill,

1998), responding maintained by water in water-deprived rats (Carli, Invernizzi,

Cervo, & Samanin, 1988), and the time spent drinking sweetened condensed milk

(Simansky & Vaidya, 1990). To our knowledge there had not been any

pharmacological studies to determine whether this decrease in fluid consumption is

due to effects at 5-HT1A or 5-HT1B receptors. In many ways the adipsic response to RU

24969 would be a preferable behavioural response to measure, because only one drug

exposure is required, and because the effect can be assessed in a relatively short time

period. Thus, the next study aimed to determine the parameters of RU 24969-produced

adipsia, and the relative contribution of 5-HT1A and 5-HT1B receptor subtypes to this

effect.

45

Chapter 4: Behavioural responses to RU 24969

Parts of this chapter appear in:

Aronsen, Webster, & Schenk. (2014). RU 24969-produced adipsia and

hyperlocomotion: Differential role of 5-HT1A and 5-HT1B receptor

mechanisms. Pharmacology, Biochemistry and Behavior, 124, 1-4. DOI

10.1016/j.pbb.2014.05.008

Aronsen, Bukholt, & Schenk (2016). Repeated administration of the 5-HT1B/1A agonist,

RU 24969, facilitates the acquisition of MDMA self-administration: Role of 5-

HT1A and 5-HT1B receptor mechanisms. Psychopharmacology, 233 (8), 1339-

1347. DOI 10.1007/s00213-016-4225-x

The previous chapter showed that latent inhibition, assessed using the

conditioned taste aversion paradigm, is a confounded behavioural assay due to

decreased fluid consumption produced by RU 24969. This decrease in fluid

consumption might, however, be a novel response that could be used to characterise

RU 24969. This effect had been referred to in the literature, but no study had

determined the parameters of this adipsic response to RU 24969, or the contribution of

5-HT1A and 5-HT1B receptors. This was, therefore, one objective of this study.

RU 24969 also produces hyperlocomotion. In contrast to RU 24969-produced

adipsia, this behavioural response to RU 24969 has been well studied. RU 24969-

produced hyperactivity was not attenuated by depletion of brain 5-HT, suggesting a

post synaptic mechanism (Cheetham & Heal, 1993). Studies in mice have generally

attributed RU 24969-induced hyperlocomotion to 5-HT1B mechanisms because it was

selectively attenuated by pretreatment with 5-HT1B, but not 5-HT1A, receptor

antagonists (Cheetham & Heal, 1993; Shanahan et al., 2009). In the rat, however, there

is a lack of full parametric analysis of the roles of 5-HT1A or 5-HT1B activation in this

behavioural response. For example, the 5-HT1B/1D receptor antagonist, GR 127935,

dose-dependently attenuated the hyperactive response to RU 24969 in the Wistar-

Kyoto hyperactive rat, but a control strain was not assessed (Chaouloff, Courvoisier,

Moisan, & Mormede, 1999). Similarly, GR 127935 blocked the hyperactive response

to RU 24969 50-60 minutes after RU 24969 administration (O’Neill & Parameswaran,

1997), while the 5-HT1A receptor antagonists, WAY 100635 and SDX 216-525, but

not GR 127935, blocked the hyperactive response to RU 24969 in the first 15 minutes

after administration (Kalkman, 1995), before maximal effects of RU 24969 are

46

evident. Thus, roles of 5-HT1A and 5-HT1B receptors in RU 24969-produced

hyperlocomotion have been suggested, but the relative contribution of 5-HT1A and 5-

HT1B receptors over the course of RU 24969-produced hyperactivity is not clear.

These studies had 3 aims. Firstly, the adipsic and hyperactive responses to RU

24969 were characterised by administering a range of doses and measuring dose-

dependent behavioural responses. Secondly, the relative contributions of 5-HT1A and

5-HT1B receptors to these effects were determined by pretreating rats with a selective

5-HT1A or 5-HT1B receptor antagonist. Lastly, if a behavioural response to RU 24969

was antagonised by a 5-HT1A receptor antagonist the same response was to be tested

after administration of the selective 5-HT1A receptor agonist, 8-OH-DPAT. A more

selective, well characterised, 5-HT1B receptor agonist was not readily available to us at

the time of these experiments, but 8-OH-DPAT has been widely used as a selective 5-

HT1A receptor agonist. 8-OH-DPAT has approximately 7000 fold preference for 5-

HT1A receptors over 5-HT1B receptors (Hamon, Cossery, Spampinato, & Gozlan,

1986). Therefore, 8-OH-DPAT is a preferable ligand to use when measuring

behavioural responses to 5-HT1A receptor activation.

Method

Water consumption

Standard protocol was used (see General Methods). RU 24969 (0.0 – 3.0

mg/kg, s.c.; n = 10 per group) was administered 15 minutes before water bottles were

reintroduced. These data provided the dose of RU 24969 that was subsequently used in

the antagonist study. Separate groups (n=6-9 per group) were tested in the same

manner to assess the contribution of 5-HT1A or 5-HT1B mechanisms. Either the 5-HT1A

receptor antagonist, WAY 100635 (0.0, 1.0 mg/kg, s.c.), or the 5-HT1B/1D receptor

antagonist, GR 127935 (0.0, 3.0 mg/kg, s.c.) was administered 15 minutes before RU

24969 (1.0 mg/kg, s.c.). These doses were chosen for their documented efficacy in

blocking 5-HT1A and 5-HT1B effects, respectively (Acosta et al., 2005; P. J. Fletcher &

Korth, 1999b).

Locomotor Activity

Rats were placed in the testing chamber for 30 minutes, followed by an

injection of RU 24969 (0.0-3.0 mg/kg, s.c.; n=8 per group), and activity was measured

for 45 minutes post-injection. Separate groups (n=6-12 per group) were placed in the

activity monitoring chambers and 15 minutes later received either WAY 100635 (0.0,

47

1.0 mg/kg, s.c.) or GR 127935 (0.0, 3.0 mg/kg, s.c.), followed 15 minutes later by RU

24969 (3.0 mg/kg, s.c.). In order for the data to be directly comparable to the fluid

consumption protocol, only data collected from 15-45 minutes following the injection

of RU 24969 were analysed.

Separate rats were used to test the hyperactive response to 8-OH-DPAT. Rats

were placed in the testing chamber for 30 minutes, followed by an injection of 8-OH-

DPAT (0.0, 0.03, 0.1, 0.3, 1.0, 3.0 mg/kg, s.c., n=5-7 per group), and activity was

measured for 60 minutes post-injection.

8-OH-DPAT is a selective 5-HT1A receptor agonist but also has appreciable

affinity for 5-HT7 receptors (Bard et al., 1993; Lovenberg et al., 1993). To determine

whether 8-OH-DAT-produced hyperactivity was due to 5-HT1A activation we

determined the effect of the selective 5-HT1A receptor antagonist, WAY 100635, on 8-

OH-DPAT-produced hyperactivity. Rats were placed in the testing chamber and 15

minutes later were injected with WAY 100635 (0, 0.003, 0.3 mg/kg, s.c., n=4-5 per

group). Following a further 15 minutes, 8-OH-DPAT (0.3 mg/kg, s.c.) was injected,

and activity was measured for an additional 60 minutes.

Data analysis

The effect of RU 24969 on water consumption was assessed using a one-way

ANOVA. The effect of RU 24969 on locomotor activity was assessed using a 4 (RU

24969 dose) × 6 (Time after injection) mixed ANOVA with Time as the within

subjects factor. The effects of WAY 100635 and GR 127935 on RU 24969-proudced

adipsia or hyperlocomotion were assessed using separate 2 (antagonist dose) × 2 (RU

24969 dose) ANOVAs. The effect of 8-OH-DPAT on locomotor activity counts was

assessed using a one-way ANOVA. Data for 8-OH-DPAT-produced hyperactivity

after administration of WAY 100635 were analysed using a 3 (Dose) × 12 (Time after

injection) mixed model ANOVA with Time as the within subjects factor. Post-hoc

analyses were conducted using Tukey’s HSD method.

48

Results

0.0 0.03 0.3 1.0 3.0

0

5

10

15

RU 24969 (mg/kg)

Wat

er c

onsu

mpt

ion

(ml)

*

*

*

Fig. 4.1 Effect  of  RU  24969  on  water  consumption  over  30  minutes  in  water  deprived  rats.  n=  10  per  group,  error  bars  represent  SEM.  *-­‐  p<0.05  compared  to  0.0  mg/kg  dose.

Figure 4.1 shows the effect of RU 24969 on water consumption. ANOVA

confirmed an effect of dose (F (4, 45) = 24.56, p<0.001, ɳp2= 0.69), and post hoc

Tukey analysis indicated that 0.3, 1.0, and 3.0 mg/kg RU 24969 significantly

decreased water consumption (p<0.05). Effects of the antagonists on RU 24969-

produced adipsia are presented in Figure 4.2

Fig. 4.2 Effect  of  the  5-­‐HT1B/1D  receptor antagonist,  GR  127935  (left),  or  the  5-­‐HT1A  receptor  antagonist,  WAY  100635  (right),  on  RU  24969-­‐produced  adipsia.  n=6-­‐9  per  group,  error  bars  represent  SEM.  *-­‐  p<0.05.  

0 .0 1 .00

5

1 0

1 5

R U 2 4 9 6 9 (m g /k g )

Wa

ter

co

ns

um

pti

on

(m

l)

0 .0

1 .0

W A Y 1 0 0 6 3 5 (m g /k g )

0 .0 1 .00

5

1 0

1 5

R U 2 4 9 6 9 (m g /k g )

Wa

ter

co

ns

um

pti

on

(m

l)

0 .0

3 .0

G R 1 2 7 9 3 5 (m g /k g )

*

49

Analysis of the effect of WAY 100635 (dose RU 24969 × dose WAY 100635)

revealed a main effect of RU 24969 (F (1,26) = 26.95, p<.001, ɳp2= 0.51), but no effect

of WAY 100635 (F (1,26) = 0.016, ns) or an interaction (F (1,26) = 0.83, ns). In

contrast, analysis of the effect of GR 127935 (dose RU 24969 × dose GR 127935)

revealed an effect of GR 127935 (F (1,24) = 4.55, p=0.043, ɳp2= 0.16), an effect of RU

24969 (F (1,24) = 29.44, p<0.001 ɳp2= 0.55) and an interaction (F (1,24) = 9.02, p =

0.006 ɳp2= 0.27). Tukey post hoc comparisons confirmed that GR 127935 significantly

reduced RU 24969-produced adipsia (p<0.05).

Figure 4.3 shows that RU 24969 increased locomotor activity (F (3,28) = 8.15,

p<0.001 ɳp2= 0.47). There was no effect of Time (F (5,140) = 0.27, ns) and no

interaction (F (15,140) = 0.45, ns). Post hoc Tukey analysis showed the dose of 3.0

mg/kg was the only dose that significantly increased total forward locomotion.

Fig. 4.3 Effect  of  RU  24969  on  locomotor  activity.  n=8  per  group,  error  bars  represent  SEM.  *-­‐  p<0.05.  

Effects of the antagonists on RU 24969-produced hyperlocomotion are

presented in figure 4.4. GR 127935 failed to alter RU 24969-produced hyperactivity;

the effect of GR 127935 (F (1,26) = 0.75, ns) and the interaction (F (1,26) = 0.52, ns)

between the two drugs were not significant. A significant effect of WAY 100635 was

found (F (1,36) = 6.73, p = 0.014, ɳp2= 0.16), and an interaction between WAY

100635 and RU 24969 treatment was significant (F (1,36) = 4.44, p = 0.042, ɳp2=

0.11). Tukey post hoc comparisons confirmed that WAY 100635 significantly reduced

RU 24969-produced hyperactivity (p<0.05).

-3 5 -3 0 -2 5 -2 0 -1 5 -1 0 -5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 50

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

T im e (m in )

Am

bu

lato

ry c

ou

nts

0 .0

0 .3

1 .0

3 .0

R U 2 4 9 6 9 (m g /k g )

R U 2 4 9 6 90 .0 0 .3 1 .0 3 .0

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

R U 2 4 9 6 9 (m g /k g )

To

tal a

mb

ula

tory

co

un

ts

*

50

Fig. 4.4 Effect  of  the  5-­‐HT1B/1D  receptor  antagonist,  GR  127935  (left),  or  the  5-­‐HT1A  receptor  antagonist,  WAY  100635  (right),  on  RU  24969-­‐produced  hyperactivity.  n=6-­‐12  per  group,  error  bars  represent  SEM.  *-­‐  p<0.05.  

8-OH-DPAT dose-dependently increased locomotor activity counts

(F(5,33)=48.63, p<0.001, ɳp2= 0.88). Post hoc analysis revealed that doses of 0.3, 1.0,

and 3.0 mg/kg 8-OH-DPAT significantly increased locomotor activity counts (see fig

4.5).

0 .00 .0

30 .1 0 .3 1 .0 3 .0

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

8 -O H -D P A T (m g /k g )

To

tal

am

bu

lato

ry c

ou

nts

**

*

Fig. 4.5 The  hyperactive  response  to  8-­‐OH-­‐DPAT.  n=5-­‐7  per  group,  error  bars  represent  SEM.  *-­‐  p<0.05  compared  to  0.0  mg/kg  group.  

Figure 4.6 (left panel) shows the time course of the effects of WAY 100635 on

8-OH-DPAT-produced hyperactivity. ANOVA showed a significant interaction

0 .0 3 .00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

3 5 0 0

R U 2 4 9 6 9 (m g /k g )

To

tal

am

bu

lato

ry c

ou

nts

0 .0

1 .0

G R 1 2 7 9 3 5 (m g /k g )

0 .0 3 .00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

3 5 0 0

R U 2 4 9 6 9 (m g /k g )

To

tal

am

bu

lato

ry c

ou

nts

0 .0

1 .0

W A Y 1 0 0 6 3 5 (m g /k g )*

51

between Time after injection and Dose (F(22,121)=7.66, p<0.001, ɳp2= 0.58), and

significant main effects of Time (F(11,121)=31.1, p<0.001, ɳp2= 0.74) and Dose

(F(2,11)=21.5, p<0.001, ɳp2= 0.80). Post hoc tests revealed a significant decrease in 8-

OH-DPAT-produced hyperactivity at Time=5, 10 and 15 minutes following

administration of 0.3 mg/kg WAY 100635. The effect of dose is further illustrated in

Figure 4.6 (right panel). Post hoc analysis showed a significant decrease in 8-OH-

DPAT-produced hyperactivity after the 0.3 mg/kg dose of WAY 100635.

-25

-20

-15

-10 -5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0

0

2 0 0

4 0 0

6 0 0

T im e (m in )

Am

bu

lato

ry c

ou

nts

0 .0 W A Y 1 0 0 6 3 5

0 .0 0 3 W A Y 1 0 0 6 3 5

0 .3 W A Y 1 0 0 6 3 5

* **

0 .0

0 .00 3

0 .30

1 0 0 0

2 0 0 0

3 0 0 0

W A Y 1 0 0 6 3 5 (m g /k g )T

ota

l a

mb

ula

tory

co

un

ts

*

Fig. 4.6 (left  panel)  Time  course  of  8-­‐OH-­‐DPAT-­‐  (0.3  mg/kg)  produced  locomotor  activity  following  WAY  100635.  (right  panel)  Effects  of  WAY  100635  on  total  locomotor  activity  following  administration  of  8-­‐OH-­‐DPAT  (0.3  mg/kg).  n  =  5-­‐6  per  group,  error  bars  represent  SEM.  *-­‐  p<0.05  compared  to  WAY  100635  0.0  mg/kg  group.   Discussion

The 5-HT1B/1A receptor agonist, RU 24969, dose dependently decreased water

consumption and increased locomotor activity. The different potencies of RU 24969 in

the two behavioural paradigms were consistent with the differential affinity of RU

24969 for the 5-HT1A and 5-HT1B receptors; RU 24969 has about 6 times greater

affinity for the 5-HT1B receptor subtype than the 5-HT1A receptor subtype (Peroutka,

1986). In the behavioural tasks, the lowest dose of RU 24969 that affected fluid

consumption was 0.3 mg/kg, while RU 24969-induced hyperlocomotion was only

observed after 3.0 mg/kg.

The most convincing evidence of different receptor mechanisms for RU 24969-

induced adipsia and hyperlocomotion is that a dose of the 5-HT1B/1D receptor

antagonist, GR 127935, which blocked the adipsic effect, failed to alter the

52

hyperlocomotion effect of RU 24969. Further, a dose of the 5-HT1A receptor

antagonist, WAY 100635, which blocked the locomotor activating effects failed to

alter the adipsic response to RU 24969. The failure of these doses of WAY 100635 and

GR 127935 to alter one behaviour cannot be due to ineffective dosing since the other

behavioural effect of RU 24969 was attenuated by the same dose of the antagonist. It

might be argued that the decrease in drinking reflects the hyperactive response to RU

24969 that might have interfered with the ability to remain at the drinking spout. This

is unlikely since the reduction of fluid consumption was produced by doses of RU

24969 lower than those that increased locomotor activity.

Rather, the data are consistent with the idea that RU 24969-induced adipsia in

rats is mediated by 5-HT1B, but not 5-HT1A, mechanisms, and that RU 24969-induced

hyperactivity in rats is mediated by 5-HT1A, but not 5-HT1B, mechanisms. Another

study (Chaouloff et al., 1999) showed that GR 127935 attenuated RU 24969-induced

hyperactivity in Wistar-Kyoto hyperactive rats. This effect might have been non-

selective since GR 127935 in that study also decreased basal activity levels. We failed

to observe either of these effects in Sprague-Dawley rats, raising the possibility that

there are strain differences in the response to the antagonist. Another study (O’Neill &

Parameswaran, 1997) also showed that RU 24969-induced hyperactivity was

decreased by GR 127935, but this effect was produced 50-60 minutes after RU 24969

administration. This finding raises the possibility that there is an effect of GR 127935

that emerges at time points later than those tested in the present study. In accordance

with our conclusion that RU 24969-induced hyperlocomotion is due to this agonist’s

affinity for the 5-HT1A receptor, 5-HT1A receptor agonists are known to produce

hyperlocomotion (Kalkman & Soar, 1990; Tricklebank, Forler, & Fozard, 1984).

GR 127935 has affinity for a number of serotonin receptors (Centurión et al.,

2000; Price et al., 1997; Watson, Burton, Price, Jones, & Middlemiss, 1996), but it is

noteworthy that the 5-HT1B/1D receptor antagonist is at least 60 times more selective

for the 5-HT1B receptor than any of the other receptors that RU 24969 has notable

affinity for. Therefore, the most likely explanation for the reversal of RU 24969-

induced adipsia by GR 127935 is antagonism of the 5-HT1B receptor.

These results, along with others from the literature, raised the possibility that a

5-HT1A receptor agonist would also produce reliable hyperlocomotion. Given the high

selectivity of 8-OH-DPAT for the 5-HT1A receptor, as well as the low affinity for the

5-HT1B receptor (Hamon et al., 1986; Peroutka, 1986), 8-OH-DPAT-produced

53

hyperactivity would be a preferable behavioural measure of 5-HT1A activation. Thus,

the last study in this chapter aimed to determine the parameters under which 8-OH-

DPAT produces hyperactivity, and the role of 5-HT1A receptor activation in this effect.

8-OH-DPAT dose dependently increased locomotor activity, with a maximal

effect around 1.0 mg/kg. This hyperactive response to the 5-HT1A receptor agonist was

reversed by the 5-HT1A receptor antagonist, WAY 100635. It is unlikely that this

reversal by WAY 100635 was due to a non-specific decrease in locomotor activity

because the higher (1.0 mg/kg) dose of WAY 100635 used in the previous experiment

had no significant effect on locomotor activity. Therefore, these results suggest that 8-

OH-DPAT-produced hyperactivity is due to 5-HT1A receptor activation.

This result is in accordance with other studies that have investigated the

hyperlocomotor response to 8-OH-DPAT. Hyperactivity produced by 8-OH-DPAT

was attenuated by the 5-HT1 receptor antagonist, pindolol (Ahlenius & Salmi, 1995;

Hillegaart, Estival, & Ahlenius, 1996), suggesting a 5-HT1A receptor mechanism. 8-

OH-DPAT produced hyperlocomotion was not attenuated by depletion of monoamines

via reserpine treatment, suggesting this behavioural response to 8-OH-DPAT is not

due to alterations in synthesis and/or release of 5-HT via autoreceptor-mediated

effects, but instead action on post-synaptic 5-HT1A receptors (Ahlenius & Salmi, 1995;

Mignon & Wolf, 2002).

Together, these data show that adipsia and hyperlocomotion provide

dissociable behavioural measures of RU 24969 that are produced by 5-HT1B and 5-

HT1A activation, respectively. Furthermore, 8-OH-DPAT-produced hyperactivity may

be a preferable measure of 5-HT1A activation, because of the selectivity of 8-OH-

DPAT for the 5-HT1A receptor. Because RU 24969-produced adipsia and 8-OH-

DPAT-produced hyperactivity are selective responses to 5-HT1B and 5-HT1A receptor

activation, respectively, these procedures provide straight-forward assays of 5-HT1A

and 5-HT1B receptor function, and so will be used in the following chapters.

54

Chapter 5: Effects of repeated administration of the 5-HT1B/1A receptor agonist,

RU 24969, on the acquisition of MDMA self-administration

Parts of this chapter appear in:

Aronsen, Bukholt, & Schenk (2016). Repeated administration of the 5-HT1B/1A agonist,

RU 24969, facilitates the acquisition of MDMA self-administration: Role of 5-

HT1A and 5-HT1B receptor mechanisms. Psychopharmacology, 233 (8), 1339-

1347. DOI 10.1007/s00213-016-4225-x

As was explained in the General Introduction, self-administration of a range of

substances is inhibited by increased synaptic 5-HT (Loh & Roberts, 1990; Ritz &

Kuhar, 1989; Rothman et al., 2005; Z. Wang & Woolverton, 2007; Wee et al., 2005).

A recent study (Bradbury et al., 2014) tested the idea that MDMA-produced 5-HT

release might be inhibitory to MDMA self-administration and attempted to explain

both the long latency to acquisition, and the small proportion of rats that meet

acquisition criteria. The MDMA-produced increase in synaptic 5-HT was measured by

in vivo microdialysis before MDMA self-administration began. As has been observed

in many studies from the Schenk lab (Colussi-Mas et al., 2010; Schenk et al., 2012;

Schenk et al., 2003; Schenk et al., 2007), about 50% of the rats acquired MDMA self-

administration. Of interest, MDMA-stimulated 5-HT release was lower for the rats that

ultimately met the acquisition criteria, suggesting an inhibitory role of MDMA-

produced 5-HT release on the acquisition of MDMA self-administration. This idea was

experimentally tested by determining the effect of a neurotoxic, 5,7-DHT, lesion on

MDMA self-administration. The lesion reduced 5-HT levels by up to 67%, and greatly

facilitated the acquisition of MDMA self-administration; while approximately 50% of

control rats met acquisition criteria, 100% of the lesion group acquired. Furthermore,

of the control group that acquired, 50% met the criterion within 14 sessions, while only

6 sessions were required for 50% of the lesion group to meet the criterion.

These findings strengthen the idea that variability in the acquisition of MDMA

self-administration is due to variability in sensitivity to MDMA-produced 5-HT

release. Specifically, 5-HT has an inhibitory impact on MDMA self-administration. A

question remains as to the mechanism for this inhibitory effect of 5-HT on the

acquisition of MDMA self-administration. One possibility is that high levels of

synaptic 5-HT produced by MDMA during initial self-administration sessions led to

55

neuroadaptive changes in 5-HT receptor mechanisms that modulate responses

associated with the acquisition of self-administration.

As outlined in the General Introduction, 5-HT1A and 5-HT1B receptors regulate

dopaminergic neurotransmission. Because self-administration is associated with

increased dopamine neurotransmission, activation of 5-HT1A and 5-HT1B receptors

might be expected to impact self-administration. Of particular interest, activation of 5-

HT1A receptors attenuated amphetamine-induced increases in extracellular dopamine

levels (Ichikawa et al., 1995; Kuroki et al., 1996) and, as would therefore be expected,

a range of 5-HT1A receptor agonists have been shown to inhibit self-administration

(Müller et al., 2007). On the other hand, 5-HT1B receptor agonists potentiated the

increase in extracellular dopamine produced by cocaine or ethanol (O'Dell & Parsons,

2004; Parsons et al., 1999; Yan et al., 2005), and generally enhanced self-

administration, producing leftward shifts in the self-administration dose-response

curves for cocaine and GBR 12909 (Parsons et al., 1996, 1998; Pentkowski et al.,

2009; Przegaliñski et al., 2007).

A wealth of data indicate a role of 5-HT1A and 5-HT1B receptor subtypes in the

maintenance of self-administration (P. J. Fletcher, Azampanah, & Korth, 2002;

Neisewander, Cheung, & Pentkowski, 2014; Parsons et al., 1998; Peltier & Schenk,

1993; Przegaliñski et al., 2007) but the role in the acquisition of self-administration has

received far less attention. Given that self-administration is driven by increases in

dopamine neurotransmission, and that the acquisition of MDMA self-administration

was enhanced by a neurotoxic, 5,7-DHT lesion, I wanted to determine whether this

facilitation of MDMA self-administration was due to decreased activation of 5-HT1A

or 5-HT1B receptors. If so, it should be possible to manipulate receptor mechanisms via

repeated agonist or antagonist exposure and to determine the effect on acquisition of

MDMA self-administration.

Tolerance to RU 24969-produced hyperactivity was produced following

repeated exposure to the 5-HT1B/1A receptor agonist (Oberlander, Demassey, Verdu,

Van de Velde, & Bardelay, 1987). As outlined in the previous chapter, we have

recently shown that RU 24969-produced hyperactivity in rats is due to activation of 5-

HT1A, but not 5-HT1B, receptors (Aronsen, Webster, & Schenk, 2014), suggesting that

behavioural tolerance reflects a down-regulation of this receptor subtype. The effect of

RU 24969 pretreatment on 5-HT1B receptor mechanisms has not been specifically

measured, but RU 24969-produced adipsia provides a means of addressing this

56

question (Aronsen et al., 2014). Therefore, in the present study we determined the

effect of repeated exposure to RU 24969 on the acquisition of MDMA self-

administration, and on RU 24969-produced adipsia. In order to assess the effect on 5-

HT1A receptor mechanisms we also measured hyperactivity in response to the selective

5-HT1A receptor agonist, 8-OH-DPAT.

Method

Male Sprague-Dawley rats (see General Methods) were used. For rats that

underwent self-administration testing, an intravenous catheter was implanted, as

outlined in the General Methods.

RU 24969 pretreatment

RU 24969 pretreatment began once pre-surgery weight had been obtained. RU

24969 (3.0 mg/kg, s.c.), or the saline vehicle (1.0 ml/kg), was administered in the

home cage daily at 0900hr and 1600hr, for three consecutive days. This protocol was

adapted from that used in earlier studies (Callaway & Geyer, 1992; Oberlander et al.,

1987) to utilise a dose of RU 24969 that we have previously shown produces both

hyperactivity and adipsia (Aronsen et al., 2014).

Acquisition of MDMA self-administration

Self-administration sessions began the day after the last administration of RU

24969. Self-administration was conducted during 2 hour daily sessions, 6 days per

week. Each self-administration session began with an experimenter-delivered infusion

of drug to clear the line of heparinised saline solution. Thereafter, depression of the

active lever produced an infusion of MDMA (1.0 mg/kg/infusion) according to an FR1

schedule. Responses on the active and inactive levers were recorded. Every seventh

day catheters were infused with sodium pentobarbital (20.0 mg/kg, i.v.). Failure to

demonstrate an immediate loss of the righting reflex suggested a loss of catheter

patency and the rat was excluded from the study. Catheter patency was lost in 4 rats (3

RU 24969 pretreated, 1 saline pretreated), and 3 rats in the RU 24969 pretreatment

group self-administered lethal doses of MDMA, resulting in final sample sizes of 9 and

8 for the RU 24969 and saline pretreated groups, respectively. Self-administration

testing continued for each rat until a total of 90 infusions (90.0 mg/kg) had been self-

administered, or for 25 days, whichever came first. This acquisition criterion is the

same as has been used previously in our laboratory (Bradbury et al., 2014; Oakly,

Brox, Schenk, & Ellenbroek, 2014).

57

Water consumption and locomotor activity

Separate groups of rats were tested to determine the effects of RU 24969

pretreatment on RU 24969-produced adipsia, or 8-OH-DPAT-produced hyperactivity.

The standard water consumption protocol was used (see General Methods), with water

bottles removed the day after the last RU 24969 pretreatment administration. RU

24969 (0.0, 1.0, 3.0 mg/kg, s.c., n= 6-8 per group) was administered 15 minutes before

water bottles were reintroduced. These doses were chosen based on our previous study

(Aronsen et al., 2014) that suggested that adipsia following administration of these

doses of RU 24969 was due to 5-HT1B receptor activation.

The effect of the selective 5-HT1A receptor agonist, 8-OH-DPAT, on locomotor

activity was assessed 2 days after the last administration of RU 24969, in order to

match the delay between pretreatment and the test for RU 24969-induced adipsia. Rats

were placed in the testing chamber (see General Methods) for 30 minutes, followed by

an injection of 8-OH-DPAT (0.0, 0.1, 0.3 mg/kg, s.c., n=4-7 per group), and activity

was measured for 60 minutes post-injection.

To investigate the possibility that RU 24969 pretreatment affected

dopaminergic mechanisms, locomotor activity produced by the dopamine releasing

agent, d-amphetamine, was assessed 2 days after the last administration of RU 24969.

Rats pretreated with either RU 24969, or vehicle, were placed in the testing chamber

(see General Methods) for 30 minutes, followed by an injection of d-amphetamine (0.5

mg/kg, i.p., n=10 per group), and activity was measured for 60 minutes post-injection.

This dose was chosen because it has previously been used in our laboratory to illustrate

dopaminergic sensitisation (Bradbury, Gittings, & Schenk, 2012).

Data analysis

Acquisition of self-administration was compared between pretreatment groups

with a survival analysis, using the log-rank test to compare Kaplan-Meier survival

estimates (Kaplan & Meier, 1958). Right-censoring was applied to data from rats that

did not acquire within the 25 day cut-off period.

RU 24969-produced adipsia was analysed with a 2 (Pretreatment) × 3 (Dose of

RU 24969) ANOVA. Effects of each dose of 8-OH-DPAT on locomotor activity were

analysed by individual 2 (Pretreatment) × 12 (Time after injection) mixed model

ANOVAs with Time as the within subjects factor. Total activity counts as a function

of Dose of 8-OH-DPAT and RU 24969 pretreatment were analysed using a 2

(Pretreatment) × 3 (8-OH-DPAT Dose) ANOVA. The locomotor responses to d-

58

amphetamine was analysed with a 2 (pretreatment) × 12 (Time after injection) mixed

model ANOVA with Time as the within subjects factor.

Results

Figure 5.1 shows survival curves for the acquisition of self-administration for

saline- or RU 24969-treated groups. RU 24969 pretreatment produced a significant

increase in the probability of acquiring MDMA self-administration (χ² (1) = 12.21,

p<0.01). Of the control group that met the acquisition criterion, 50% met the criterion

within 17 sessions, whereas 50% of RU 24969 pretreatment group met the acquisition

criterion within 10 sessions. It is noteworthy that three rats in the RU 24969

pretreatment group self-administered lethal doses of MDMA (>20 mg/kg) during the

first self-administration session and therefore additional data from these rats could not

be obtained. The high intake during the first self-administration session for these 3 rats

supports the other data suggesting RU 24969 pretreatment enhanced the initial

reinforcing effects of MDMA.

0 5 10 15 20 250

25

50

75

100

Self-administration session

Perc

ent a

cqui

red

Saline PretreatmentRU 24969 pretreatment

Fig. 5.1 Cumulative  percentage  of  rats  that  met  the  criterion  for  acquisition  of  MDMA  self-­‐administration  in  the  RU  24969  (squares,  n=9)  and  saline  (circles,  n=8)  pretreatment  groups.  

Figure 5.2 (left panel) shows the effect of RU 24969 pretreatment on RU

24969-produced adipsia. There was a significant interaction between Pretreatment and

Dose (F(2,37)=7.85, p=0.01, ɳp2= 0.30) and a significant effect of Dose

(F(2,37)=53.55, p<0.01, ɳp2= 0.74). Post hoc tests confirmed a significant difference in

59

the adipsic response between the RU 24969 and saline pretreatment groups following

0.0 mg/kg, and 3.0 mg/kg RU 24969. Since there was a decrease in basal water

consumption produced by repeated RU 24969 treatment, the data were further

analysed by expressing drug effects as a percentage of baseline. These data are

presented in Figure 5.2 (right panel). A 2×2 (Pretreatment × Dose) ANOVA revealed a

significant effect of Pretreatment (F(1,27)=20.40, p<0.01, ɳp2= 0.43).

0.0

1.0 3.00

5

10

15

RU 24969 (mg/kg)

Wat

er c

onsu

mpt

ion

(ml)

Saline PretreatmentRU 24969 pretreatment

*

*

1.0 3.00

20

40

60

80

100

RU 24969 (mg/kg)

Wat

er c

onsu

mpt

ion

- P

erce

ntag

e of

bas

elin

e

Fig. 5.2 (Left  panel)  The  adipsic  response  to  RU  24969  after  repeated  exposure  to  RU  24969  (grey  bars)  or  saline  (black  bars).  (Right  panel)  Percentage  of  baseline  water  intake  as  a  function  of  RU  24969  dose  for  RU  24969  and  saline  pretreated  groups.  n=6-­‐8  per  group.  Figures  represent  the  mean  +  SEM.  *  -­‐  p<0.05.  

Locomotor activity produced by the various doses of 8-OH-DPAT as a

function of RU 24969 pretreatment is shown in Figure 5.3. There were no differences

between groups following the 0.0 mg/kg 8-OH-DPAT dose. The data from 0.1 mg/kg

8-OH-DPAT dose produced a Time × Pretreatment interaction (F(11,110)=4.06,

p<0.01, ɳp2= 0.29) and main effects of Time (F(11,110)=19.8, p<0.01, ɳp

2= 0.66) and

Pretreatment (F(1,10)=17.6, p<0.01, ɳp2= 0.64). Post hoc tests revealed significant

decreases in activity during Time=10 and 15 minutes following the injection. There

was a significant Time × Pretreatment interaction (F(11,121)=2.77, p<0.01, ɳp2= 0.20)

and main effects of Time (F(11,121)=62.5, p<0.01, ɳp2= 0.85) and Pretreatment

(F(1,11)=7.45, p<0.05, ɳp2= 0.40) for the 0.3 mg/kg 8-OH-DPAT groups. Post-hoc

tests revealed a significant decrease in activity at Time=25 minutes. Analysis of total

activity counts as a function of Dose and Pretreatment showed a main effect of 8-OH-

60

DPAT Dose (F(2,27)=46.0, p<0.01, ɳp2= 0.77) and a main effect of Pretreatment

F(1,27)=19.5, p<0.01, ɳp2= 0.42).

-25

-20

-15

-10 -5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0

0

2 0 0

4 0 0

6 0 0

0 .0 m g /k g

T im e (m in )

Am

bu

lato

ry c

ou

nts

- 25

-20

-15

-10 -5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0

0

2 0 0

4 0 0

6 0 0

0 .1 m g /k g

T im e (m in )

Am

bu

lato

ry c

ou

nts

*

*

-25

-20

-15

-10 -5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0

0

2 0 0

4 0 0

6 0 0

0 .3 m g /k g

T im e (m in )

Am

bu

lato

ry c

ou

nts

S a lin e p re tre a tm e n t

R U 2 4 9 6 9 p re tre a tm e n t

*

0 .0

0 .1 0 .30

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

T o ta l a c tiv ity

8 -O H -D P A T (m g /k g )

To

tal

am

bu

lato

ry c

ou

nts

S a lin e P re tre a tm e n t

R U 2 4 9 6 9 p re tre a tm e n t

Fig 5.3. Locomotor  activating  effects  of  8-­‐OH-­‐DPAT  (Top  left  –  0  mg/kg,  top  right  –  0.1  mg/kg,  bottom  left  –  0.3  mg/kg,  bottom  right  –  totals)  as  a  function  of  RU  24969  or  saline  pretreatment.  n  =  4-­‐7  per  group.  Symbols  represent  the  mean  +  SEM.  *-­‐  p<0.05.  

During testing of the hyperactive response to d-amphetamine, one rat in the RU

24969 pretreatment group jumped out of the locomotor activity chamber during

testing, and so was excluded from analyses. The final sample size for this group was

therefore 9. Figure 5.4 (left panel) shows the locomotor response to d-amphetamine for

both pretreatment groups over time. ANOVA showed no significant effect of

Pretreatment (F(1,17)=0.19, p=0.67) and no interaction between Pretreatment and

Time (F(11,187)=0.29, p=0.99). Total locomotor activity counts after d-amphetamine

injection are shown in figure 5.4 (right panel). As indicated in the previous analysis of

variance, there was no effect of pretreatment.

61

T im e (m in )

Am

bu

lato

ry c

ou

nts

- 25

-20

-15

-10 -5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0

0

2 0 0

4 0 0

6 0 0

S a lin e P re tre a tm e n t

R U 2 4 9 6 9 p re tre a tm e n t

P re tre a tm e n t

To

tal

am

bu

lato

ry c

ou

nts

S a line

R U 24 9 6 9

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

Fig. 5.4 The  locomotor  response  to  d-­‐amphetamine  (0.5  mg/kg)  after  pretreatment  with  either  RU  24969  or  saline.  n=9-­‐10  per  group.  Discussion

Pretreatment with RU 24969 decreased the latency to acquisition of MDMA

self-administration, and increased the proportion of rats that acquired MDMA self-

administration. The leftward shift in the acquisition curve for self-administration might

reflect a sensitised reinforcing effect since higher doses of drug have also been shown

to decrease the latency to acquisition of self-administration (Carroll & Lac, 1997;

Schenk & Partridge, 2000).

A remarkable consequence of pretreatment with RU 24969 was the substantial

increase in the proportion of rats that met the criterion for acquisition of MDMA self-

administration. As we have previously reported (Bradbury et al., 2014; Schenk et al.,

2012), 50% of control rats met the criterion within the 25 day cut-off period. Thus,

some rats appear to be inherently more or less sensitive to the reinforcing effects of

MDMA. Following RU 24969 pretreatment, however, all of the rats met the criterion

for acquisition of MDMA self-administration within the limits of the study (25 test

sessions). We have suggested that the initial resistance to self-administration can be

overcome by limiting the impact of 5-HT since a similar increase in the percentage of

subjects that acquired MDMA self-administration was produced following neurotoxic

5,7-DHT lesions in rats (Bradbury et al., 2014) and in 5-HT transporter knock-out rats

(Oakly et al., 2014).

In order to assess the impact of more specific 5-HT mechanisms on the

acquisition of MDMA self-administration, the present study repeatedly administered

the 5-HT1B/1A receptor agonist, RU 24969, as a pretreatment in an attempt to down-

62

regulate 5-HT1A and 5-HT1B receptors. We determined effects of the pretreatment by

measuring behavioural responses that have been attributed to either 5-HT1B (RU

24969-produced adipsia (Aronsen et al., 2014)) or 5-HT1A (8-OH-DPAT-produced

hyperactivity (Hillegaart et al., 1996)) mechanisms.

As previously reported (Aronsen et al., 2014), RU 24969 produced dose-

dependent adipsia. The dose-response curve for this response is relatively narrow;

minimal effects were produced following administration of 0.3 mg/kg and maximal

effects were produced following administration of 3.0 mg/kg (Aronsen et al., 2014).

RU 24969 pretreatment decreased basal water consumption and when this was

accounted for, RU 24969 pretreatment decreased the subsequent RU 24969-produced

adipsic response. These findings are consistent with a rightward shift in the dose-

response curve and suggest a down-regulation of 5-HT1B receptors. 5-HT1B receptor

down-regulation has previously been evidenced by decreased mRNA levels

(Chennaoui et al., 2001; Hiroi & Neumaier, 2009) or decreased binding density

(Kindlundh et al., 2003; Suzuki, Han, & Lucas, 2010), both of which could explain the

present behavioural data.

RU 24969 pretreatment also shifted the dose-response curve for 8-OH-DPAT-

produced hyperactivity to the right; the most pronounced effect of pretreatment was on

hyperactivity produced by the lowest does of 8-OH-DPAT tested. This might explain

why a similar pretreatment with RU 24969 failed to alter hyperactivity produced by a

higher dose of 1.25 mg/kg 8-OH-DPAT (Oberlander et al., 1987).

Although 8-OH-DPAT has appreciable affinity for the 5-HT7 receptor (Bard et

al., 1993; Lovenberg et al., 1993), results from the previous chapter showed that

hyperactivity produced by 8-OH-DPAT was attenuated by the selective receptor

antagonist, WAY 100635, confirming a 5-HT1A receptor mechanism. Of interest, a

similar RU 24969 pretreatment regimen also reduced the locomotor response to RU

24969 (Callaway & Geyer, 1992), a behavioural response that we have attributed to 5-

HT1A receptor activation (Aronsen et al., 2014). Therefore, these findings are

consistent with a down-regulation of 5-HT1A receptors following RU 24969

pretreatment. 5-HT1A down-regulation has been shown via decreased agonist-

stimulated binding of [35S]GTPγS to G proteins (Fuss et al., 2013; Hensler, Vogt, &

Gass, 2010), decreased receptor binding densities or immunoreactivity (Fuss et al.,

2013; Gui et al., 2011), decreased 5-HT1A mRNA (S. Wang et al., 2009), and

63

decreased protein levels (Iyo et al., 2009; S. Wang et al., 2009). It would be of great

interest to determine which, if any, of these mechanisms can explain the present data.

The available literature is consistent with the idea that MDMA self-

administration, like self-administration of other drugs of abuse, progresses as a result

of sensitised dopamine and desensitised 5-HT responses. Thus, repeated exposure to

MDMA increased dopamine (Colussi-Mas et al., 2010; Kalivas, Duffy, & White,

1998) and decreased 5-HT (Baumann, Clark, Franken, et al., 2008; Reveron et al.,

2010; Shankaran & Gudelsky, 1999) synaptic output, as measured by in vivo

microdialysis, dopamine antagonists reduced MDMA self-administration (Brennan,

Carati, Lea, Fitzmaurice, & Schenk, 2009; Daniela, Brennan, Gittings, Hely, &

Schenk, 2004), and dopamine, but not 5-HT, agonists potentiated drug-seeking

following extinction of MDMA self-administration (Schenk, Gittings, & Colussi-Mas,

2011).

MDMA preferentially releases 5-HT and the ensuing activation of post-

synaptic receptors impacts dopamine release, providing potential mechanisms for the

enhanced dopamine response. In this study, both 5-HT1B and 5-HT1A receptor

mechanisms were down regulated, as measured by behavioural assays. Given the

selectivity of RU 24969 for 5-HT1A/1B receptors it is unlikely that alterations in a

different receptor mechanism underlies the facilitated acquisition of self-administration

found in the present study.

Because activation of 5-HT1B receptors enhanced extracellular dopamine

concentrations (Galloway et al., 1993; Hållbus et al., 1997; Iyer & Bradberry, 1996;

O'Dell & Parsons, 2004; Yan & Yan, 2001a; Yan et al., 2004) it is possible that

repeated administration of RU 24969 sensitised dopamine neurons independently of

the effect on 5-HT1A and 5-HT1B receptors. A sensitised dopamine response to MDMA

would be expected to facilitate the acquisition of MDMA self-administration. This

seems unlikely, however, because RU 24969 pretreatment had no effect on

amphetamine-produced hyperactivity. Although the amphetamine dose was chosen

based on other sensitisation studies, it is possible that a sensitised dopamine response

would have been observed if higher doses of amphetamine had been tested.

Activation of 5-HT1B receptors enhanced basal dopamine neurotransmission

(Alex & Pehek, 2007) and the dopaminergic response to drugs of abuse (O'Dell &

Parsons, 2004; Parsons et al., 1999; Yan et al., 2005), so the down-regulation of these

receptor mechanisms, which would be expected to decrease MDMA-produced

64

dopamine, cannot easily explain the facilitated self-administration. On the other hand,

a wealth of data suggest that activation of 5-HT1A receptors is inhibitory to cocaine

self-administration (Müller et al., 2007), possibly via inhibition of dopamine release

(Ichikawa & Meltzer, 2000). Therefore, a down-regulation of this receptor subtype

might be expected to disinhibit MDMA-produced dopamine, leading to more rapid

acquisition of self-administration due to increased reinforcing effects. This might also

explain the facilitated acquisition of MDMA self-administration in serotonin

transporter knockout rats (Oakly et al., 2014), since this manipulation also desensitised

5-HT1A receptor mechanisms (Homberg et al., 2008).

5-HT1A receptors are widely localised in brain and are well-positioned to

modulate activity in a large number of brain systems (Aznar, Qian, Shah, Rahbek, &

Knudsen, 2003). Of importance, these receptors are localised on tyrosine hydroxylase

immunoreactive cells in the VTA (Doherty & Pickel, 2001) and also in dopamine

terminal regions in the NAc (Alex & Pehek, 2007). Systemic administration of 8-OH-

DPAT inhibited amphetamine-produced dopamine release in the NAc (Ichikawa et al.,

1995). The down-regulation produced by RU 24969 pretreatment would, therefore, be

expected to disinhibit stimulated dopamine. Similar studies have not been conducted

using MDMA, but this mechanism could explain the facilitated acquisition of self-

administration.

The acquisition of self-administration is also influenced by factors in addition

to the initial reinforcing effects of the drug and some of these factors are modified by

5-HT1A receptor mechanisms. As explained in the General Introduction, increased

impulsivity, anxiety, or learning, could be expected to facilitate the acquisition of

MDMA self-administration.

5-HT1A activation increased behavioural measures of impulsivity (Carli &

Samanin, 2000). However, individual variability in impulsivity did not predict latency

to acquisition of MDMA self-administration (Bird & Schenk, 2013). This might be

because the impulsive response to 5-HT1A receptor agonists is due to autoreceptor

activation (Carli et al., 2006; Lladó-­‐Pelfort et al., 2010; Winstanley et al., 2003). We

have previously shown that repeated exposure to MDMA failed to alter 5-HT1A

autoreceptor mechanisms (Schenk et al., 2013). Therefore, alterations in 5-HT1A-

mediated impulsivity are unlikely to have impacted the present results.

A down-regulation of 5-HT1A receptors would be expected to reduce anxiety

produced by MDMA, because 5-HT1A receptor activation is anxiogenic (Cheeta et al.,

65

2000a, 2000b; File et al., 1996; File et al., 2000; Solati et al., 2011). However, higher

levels of anxiety have been associated with self-administration (Dilleen et al., 2012;

Homberg et al., 2002; Spanagel et al., 1995). Therefore an attenuation of 5-HT1A

receptor-produced anxiety would not explain the facilitated acquisition of MDMA self-

administration.

Reliable self-administration is often facilitated via Pavlovian conditioning

processes by pairing delivery of the drug reinforcer with a discrete, discriminative

stimulus, like a light, as was done in the present study (Di Ciano & Everitt, 2004). As

explained in the General Introduction, strengthening of stimulus/reward associations is

markedly inhibited by administration of 5-HT1A receptor agonists (Blair et al., 2004;

Frick et al., 2015; Winsauer et al., 1999). These findings raise the possibility that

activation of post synaptic 5-HT1A receptors pursuant to MDMA-stimulated 5-HT

release limits the acquisition of MDMA self-administration, in some subjects, by

interfering with associative learning. If so, our data suggest that this effect is mitigated

by exposure to a regimen of RU 24969 pretreatment that down-regulated these

receptor mechanisms, thereby facilitating MDMA self-administration as indicated by

both a leftward and upward shift in the self-administration acquisition curves. This

idea could be tested by administering the same RU 24969 pretreatment as was used in

this study and assessing learning in a stimulus/reward association task. If RU 24969

pretreatment facilitated learning in such a task it would strengthen the claim that the

facilitation of MDMA self-administration seen in the present study was associated

with enhanced learning.

66

Chapter 6: Predicting the acquisition of MDMA self-administration

In the previous chapter, a manipulation that down-regulated 5-HT1A and 5-

HT1B receptors also greatly facilitated the acquisition of MDMA self-administration.

As outlined in the discussion, it is possible that a down-regulation of these receptors

could explain the facilitated acquisition. However, the correlational nature of that

study makes it impossible to ascertain the role of alterations in 5-HT1A and/or 5-HT1B

receptor mechanisms in the acquisition of MDMA self-administration.

There is substantial evidence that the magnitude of MDMA-produced 5-HT

release predicts the latency to acquire MDMA self-administration. A question remains

as to what the mechanism underlying this effect might be. The results from the

previous chapter raise the possibility that this mechanism involves individual

variability in 5-HT1A and/or 5-HT1B receptor-mediated effects.

Some evidence suggests that activation of these receptor subtypes modulates

dopamine neurotransmission, providing a potential mechanism. For example, the 5-

HT1A receptor gonist, 8-OH-DPAT, inhibited amphetamine-produced increases in

extracellular dopamine (Ichikawa et al., 1995; Kuroki et al., 1996). Thus, activation of

5-HT1A receptors by MDMA-stimulated 5-HT would be expected to inhibit dopamine

release. If so, this might explain why MDMA is, at least initially, not a very

efficacious reinforcer. Activation of 5-HT1A receptors also impaired learning in a range

of operant tasks (Blair et al., 2004; Frick et al., 2015; Meneses, 2007; Winsauer et al.,

1999). As a result, 5-HT1A activation during MDMA self-administration could inhibit

learning processes associated with the acquisition of self-administration. Therefore,

subjects with higher sensitivity to 5-HT1A receptor activation may be less likely to

acquire MDMA self-administration, due to inhibition of dopamine release, and/or

impaired ability to learn the operant task. The observation that RU 24969 pretreatment

enhanced MDMA self-administration and down-regulated 5-HT1A receptors is

consistent with this idea.

5-HT1B receptor activation, on the other hand, augmented the increases in

extracellular dopamine produced by cocaine (O'Dell & Parsons, 2004; Parsons et al.,

1999) or ethanol (Yan et al., 2005). Thus, the down-regulation of 5-HT1B receptors

produced by RU 24969 pretreatment might be expected to reduce the reinforcing

efficacy of MDMA. Furthermore, intra-raphe injections of the neurotoxin, 5,7-DHT,

increased 5-HT1B receptor binding in the substantia nigra and NAc (Compan, Segu,

67

Buhot, & Daszuta, 1998), and intraventricular infusion of 5,7-DHT produced an

increase in 5-HT1B binding in the hypothalamus, entorhinal cortex, and substantia

nigra (Manrique et al., 1998; Manrique et al., 1994; Manrique, Segu, Hery, Faudon, &

François-Bellan, 1993; Weissmann, Mach, Oberlander, Demassey, & Pujol, 1986).

This same lesion facilitated MDMA self-administration (Bradbury et al., 2014). In

contrast, intraventricular infusion of 5,7-DHT had no impact on 5-HT1A binding in

substantia nigra, PFC, hippocampus, hypothalamus, or amygdala (Hensler, Kovachich,

& Frazer, 1991; Lawrence, Olverman, Shirakawa, Kelly, & Butcher, 1993; Weissmann

et al., 1986). Thus, the role of 5-HT1B receptor populations in MDMA self-

administration is not clear. Acquisition was facilitated by separate manipulations that

produced both an up- and down-regulation of 5-HT1B receptors, respectively. It is

therefore possible that the acquisition of MDMA self-administration is not related to 5-

HT1B receptor populations.

The purpose of the following studies was to determine whether individual

variability in 5-HT1A and/or 5-HT1B receptors predicted the latency to acquisition of

MDMA self-administration. To this end, the behavioural responses to the 5-HT1A

receptor agonist, 8-OH-DPAT, or the 5-HT1B/1A receptor agonist, RU 24969, were

assessed before MDMA self-administration commenced. Furthermore, to test the idea

that RU 24969 pretreatment facilitated acquisition of MDMA self-administration via a

down-regulation of 5-HT1A receptors, separate groups of rats were administered the 5-

HT1A receptor antagonist, WAY 100635, or vehicle, before each self-administration

session. If 5-HT1A receptor activation does inhibit MDMA self-administration,

pretreatment with WAY 100635 would be expected to facilitate acquisition.

Method

Subjects and procedures

Male Sprague-Dawley rats underwent catheter surgery for self-administration,

as outlined in the General Methods section. Testing began after recovery to pre-

surgery weight.

The hyperlocomotor response to 8-OH-DPAT was assessed using the standard

locomotor activity methods outlined in the General Methods section. Rats were placed

in the activity chambers for 30 minutes, followed by 8-OH-DPAT (0.1, 0.3 mg/kg, s.c.,

n=24 and 30 respectively) administration, and locomotor activity was measured for

another 60 minutes. These doses were chosen because inspection of preliminary data

68

showed both doses produced hyperactivity with considerable between-subject

variability.

Adipsia produced by RU 24969 was assessed using the standard water

consumption methods outlined in the General Methods section. RU 24969 (1.0 mg/kg,

s.c., n=13) was administered 15 minutes before water bottles were reintroduced. This

dose was chosen because in previous studies it produced an adipsic response with

considerable between-subject variability (Aronsen et al., 2014).

MDMA self-administration, as outlined in the General Methods, began the day

after the behavioural response to 8-OH-DPAT or RU 24969 was measured. Self-

administration was conducted during 2 hour daily sessions, 6 days per week. Each self-

administration session began with an experimenter-delivered infusion of drug.

Thereafter, depression of the active lever produced an infusion of MDMA according to

an FR1 schedule. Responses on the active and inactive levers were recorded. Every

seventh day catheters were infused with sodium pentobarbital (20.0 mg/kg, i.v.).

Failure to demonstrate an immediate loss of the righting reflex suggested a loss of

catheter patency and the rat was excluded from the study. Catheter patency was lost in

5 rats (4 after 8-OH-DPAT-produced hyperactivity (3 in the 0.1 mg/kg group, 1 in the

0.3 mg/kg group), 1 after RU 24969-produced adipsia), and one rat self-administered a

lethal dose of MDMA on the first day (0.3 mg/kg 8-OH-DPAT group). The same

acquisition criterion was used as in the last chapter – a total of 90 infusions (90 mg/kg)

self-administered. In order to minimise the number of subjects required, testing

continued for 35 sessions in the groups assigned to 0.3 mg/kg 8-OH-DPAT and 1.0

mg/kg RU 24969. Testing in the 0.1 mg/kg 8-OH-DPAT group continued for 25

sessions, whereupon subjects were used for a different study.

Using the same self-administration procedure, separate groups of rats were

pretreated with either saline vehicle, or the 5-HT1A receptor antagonist, WAY 100635

(1.0 mg/kg, s.c., n=7 per group) 15 minutes before each daily self-administration

session. Of the 14 rats that started self-administration, 4 were removed from the

experiment due to loss of catheter patency (3 in the vehicle group, 1 in the WAY

100635 group).

Statistical analyses

Behavioural responses to either 8-OH-DPAT or RU 24969 were correlated

with latency to acquisition of MDMA self-administration using a Pearson's product-

moment correlation. Data from subjects that did not acquire within the cut off period

69

were not included in these analyses. Analysis of the effect of WAY 100635

pretreatment on self-administration was not possible due to a high attrition rate, but

raw data are presented.

Results

Out of a total of 67 rats that started the locomotor and adipsia studies, 39 met

the acquisition criterion within 25 sessions, and a further 9 met the criterion between

26 and 35 sessions.

Figure 6.1 shows the distribution of days to meet the acquisition criterion and

locomotor response to 0.1 mg/kg 8-OH-DPAT. There was no significant correlation

between these two variables (r(16)=-0.21, p=0.40).

Fig 6.1 Scatterplot  of  days  to  acquire  MDMA  self-­‐administration  (y-­‐axis)  and  locomotor  response  to  0.1  mg/kg  8-­‐OH-­‐DPAT  (x-­‐axis).    

Similarly, there was no correlation between days to acquisition and locomotor

response to 0.3 mg/kg 8-OH-DPAT (r(18)=0.004, p=0.99) (see figure 6.2).

L o c o m o to r a c t iv ity c o u n ts

Da

ys

to

ac

qu

isit

ion

0 5 0 0 1 0 0 0 1 5 0 00

1 0

2 0

3 0

70

L o c o m o to r a c t iv ity c o u n ts

Da

ys

to

ac

qu

isit

ion

0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 00

1 0

2 0

3 0

4 0

Fig 6.2 Scatterplot  of  days  to  acquire  MDMA  self-­‐administration  (y-­‐axis)  and  locomotor  response  to  0.3  mg/kg  8-­‐OH-­‐DPAT  (x-­‐axis).     The distribution of days to acquire MDMA self-administration and the adipsic

response to RU 24969 is shown in figure 6.3. Analysis showed no significant

correlation between the two variables (r(7)=0.26, p=0.49).

W a te r c o n s u m p tio n

Da

ys

to

ac

qu

isit

ion

0 2 4 6 8 1 00

1 0

2 0

3 0

4 0

Fig 6.3 Scatterplot  of  days  to  acquire  MDMA  self-­‐administration  (y-­‐axis)  and  adipsic  response  to  1.0  mg/kg  RU  24969  (x-­‐axis).     Data from the WAY 100635 pretreatment groups would have been analysed

using a log-rank test to compare Kaplan-Meier survival estimates (Kaplan & Meier,

1958), but given the high attrition rate in the control group this analysis would not be

meaningful. Nonetheless the data obtained were interesting, so average self-

71

administration data over sessions are presented in figure 6.4. It is interesting to point

out that, while escalation of intake is evident from around day 6 in the control group,

there is no escalation in the WAY 100635 group. This pattern continued beyond day

15, in fact by day 25 the highest total intake in the WAY 100635 pretreatment group

was 41 mg/kg. The high variability in the vehicle control group from day 10 is to be

expected, because as we have previously shown, intake in some rats increases around

this time point (Schenk et al., 2012). Data after day 15 are not presented because, by

that stage, only 3 rats remained in the control group (1 reached acquisition criteria, 3

lost catheter patency). Table 6.1 shows the raw data for the number of infusions over

different self-administration sessions. These data further illustrate the variability in the

vehicle group, due to increased self-administration in some subjects, and is roughly in

line with the expectation that approximately 50% of control subjects would acquire

MDMA self-administration.

S e lf-a d m in is tra tio n s e s s io n

Nu

mb

er

of

infu

sio

ns

0 5 1 0 1 50

2

4

6

8

1 0W A Y 1 0 0 6 3 5 (1 .0 m g /k g )

V e h ic le

Fig 6.4 Number  of  MDMA  infusions  self-­‐administered  across  sessions  in  rats  treated  with  either  WAY  100635  (1.0  mg/kg)  or  vehicle,  15  minutes  before  self-­‐administration  commenced.  Error  bars  represent  SEM.  n=4-­‐7  per  data  point,  see  table  6.1  for  more  detail.    

72

Group Rat

Session 1 Session 5 Session 10 Session 15

WAY 100635

Pūkeko

1 0 removed removed

Rock Wren 4 0 3 1

Saddleback 2 0 0 1

Silvereye

12 0 0 1

Spotless Crake 0 1 0 0

Spotted Shag 7 1 1 0

Stichbird

1 1 2 1

Vehicle

Oystercatcher 1 0 14 removed

Takahē

2 1 2 2

Tomtit

2 1 11 15

Tūī

1 0 4 removed

Weka

1 0 0 0

White Heron 2 3 removed removed

Yellowhead 2 0 1 0

Table 6.1  The  number  of  MDMA  infusions  (1.0  mg/kg/infusion)  self-­‐administered  by  subjects  treated  with  either  the  5-­‐HT1A    receptor  antagonist,  WAY  100635,  or  saline  vehicle,  15  minutes  before  each  self-­‐administration  session.  

Discussion

These studies failed to show an association between behavioural response to 5-

HT1A or 5-HT1B activation, and latency to acquire MDMA self-administration. These

results were surprising given that, in the previous chapter, a treatment that down-

regulated both receptor subtypes also facilitated acquisition of MDMA self-

administration. Furthermore, lower sensitivity of 5-HT1A, or greater sensitivity of 5-

HT1B, receptors would be expected to enhance the dopaminergic response to MDMA,

which would be expected to enhance self-administration. Thus, it appears that basal

variability in 5-HT1A and 5-HT1B receptors is not associated with the variability in

acquisition of MDMA self-administration.

These results might suggest that facilitated self-administration after RU 24969

pretreatment reported in the previous chapter was not due to the effects of the

pretreatment on 5-HT1A or 5-HT1B receptors. Indeed, a significant correlation between

behavioural response and latency to acquisition in the present studies would have been

evidence for a role of 5-HT1A and/or 5-HT1B receptors in the initial reinforcing effects

of MDMA. However, caution should be exercised before we conclude that the effect

73

of RU 24969 pretreatment on MDMA self-administration was independent of the

effects on 5-HT1A or 5-HT1B receptors. Firstly, RU 24969 is reasonably selective for

these two receptor subtypes, making a non-selective effect less likely. Furthermore, it

is possible that the natural variability in the behaviours measured in this study was not

substantial enough to show an effect. For example, the mean activity count after 0.1

mg/kg 8-OH-DPAT in the present study was 1003 (SD=348), but after RU 24969

pretreatment this mean was 378 (SD=164). Thus, it is possible that lower sensitivity to

8-OH-DPAT is indeed predictive of latency to acquire MDMA self-administration, but

that significantly lower levels of sensitivity are required.

Further study is required to determine the relative roles of 5-HT1A and 5-HT1B

receptors in the facilitation of MDMA self-administration after RU 24969

pretreatment. Pretreatment with RU 24969 and either the 5-HT1A receptor antagonist,

WAY 100635, or the 5-HT1B receptor antagonist, GR 127935, would help to clarify

the roles of each receptor.

There was an unfortunately high attrition rate in the WAY 100635 pretreatment

study. This attrition rate likely reflects a procedural problem during catheter surgery

that has since been identified. Measures have now been put in place to avoid such

levels of attrition in future. The subsequently small size of the saline pretreatment

group precludes meaningful comparisons between the WAY 100635 pretreatment

group and its appropriate control. However, our laboratory, and this thesis, have shown

that approximately 50% of rats acquire MDMA self-administration (Bradbury et al.,

2014; Schenk et al., 2012), and it seems unlikely that saline administration would alter

this acquisition rate. Therefore, it becomes interesting that responding for MDMA was

so low in the WAY 100635 group. If these findings were replicated in a larger sample,

and with an appropriate control, the data would provide evidence for the suggestion

that 5-HT1A receptor activation is required for the development of MDMA self-

administration.

If so, it would be difficult to reconcile these data with the RU 24969

pretreatment data that showed a down-regulation of 5-HT1A receptors was associated

with enhanced MDMA self-administration. Given the limited scope of the present

study, it is not possible to rule out a non-specific effect of WAY 100635. Data from

chapter 4 suggest that this dose of WAY 100635 does not supress locomotor

responding. Furthermore, data from the first self-administration session, and other

research from our lab (Schenk et al., Under Review), show that rats are able to perform

74

an operant response after acute WAY 100635 administration. Thus it seems unlikely

that the low levels of responding after WAY 100635 administration were due to motor

effects. There were no differences between the weights of subjects in the two groups

throughout the experiment (data not shown), suggesting there was no effect of WAY

100635 on eating or drinking.

It might be expected that repeated administration of an antagonist would

upregulate receptor populations. Indeed, administration of a high dose (3 mg/kg) of

WAY 100635 twice per day for 3 days increased 5-HT1A immunoreactivity in the

hippocampus and cortex (Abbas, Nogueira, & Azmitia, 2007). An up-regulation of 5-

HT1A receptors might inhibit self-administration via enhanced inhibition of dopamine.

Alternatively, WAY 100635 may have protected 5-HT1A receptors from important

neuroadaptations in response to high levels of 5-HT. During self-administration

session 1 I noticed that the rats in the WAY 100635 group that self-administered a

large dose of MDMA did not show the characteristic set of symptoms (hyperthermia,

wetness, ‘eagle-fear’, bleeding nose) typically associated with initial self-

administration of high doses. Anecdotally, repeated self-administration of high doses

of MDMA produces tolerance to these effects. Thus, WAY 100635 may have been

preventing neuroadaptations that produce tolerance to some of the aversive effects of

MDMA. Clearly, more research would be required to determine if this is the case.

The results of the present study failed to show an association between basal

responses to 5-HT1A or 5-HT1B receptor activation and latency to acquire MDMA self-

administration. However, the results of the previous chapter, and inferences drawn

from the WAY 100635 study in this chapter, raise the possibility that neuroadaptations

in 5-HT1A and/or 5-HT1B receptors are important for the progression of MDMA self-

administration. As outlined above, basal variability in these receptor subtypes may not

be substantial enough to allow for meaningful analysis. On the other hand, if changes

in these receptor subtypes underlie the development of MDMA as an efficacious

reinforcer, it might be possible to detect differences in these receptor populations after

substantial MDMA self-administration. This possibility will be addressed in the next

chapter.

75

Chapter 7: Response to 5-HT1A and 5-HT1B receptor agonists after self-

administration

Parts of this chapter have been adapted from:

Aronsen & Schenk (2016). MDMA self-administration fails to alter the response to 5-

HT1A and 5-HT1B agonists. Psychopharmacology, 233 (7), 1323-1330. DOI

10.1007/s00213-016-4226-9

with permission from the publisher (Appendix A).

Some users regularly consume large quantities of ecstasy (Cottler et al., 2001;

Degenhardt, Barker, & Topp, 2004; Topp, Hall, & Hando, 1997), and repeated ecstasy

use produces a range of negative consequences, including cognitive and emotional

deficits. While these deficits are worrisome in and of themselves, it has been suggested

that they could also facilitate further ecstasy taking, and thus contribute to the

development of an SUD (Schenk, 2009; Schenk & Aronsen, 2015). The mechanisms

underlying these deficits are not, however, well understood.

Ecstasy users showed deficits in learning (Wagner et al., 2013), and in attention

and memory (McCann, Mertl, et al., 1999) compared to ecstasy-naïve controls or those

with limited ecstasy use. Ecstasy users reported higher levels of depression,

impulsiveness, and sleep disturbances than poly-drug users who did not use ecstasy

(Taurah et al., 2014). These cognitive and behavioural deficits were persistent,

suggesting that regular ecstasy use may cause long-lasting neuroadaptations (Parrott,

2013b).

Animal studies have shown that a number of these adverse effects associated

with ecstasy use are modulated by pharmacological manipulation of 5-HT receptors.

For example, the 5-HT1A receptor agonist, 8-OH-DPAT, impaired learning and

memory in water maze (Carli & Samanin, 1992), passive avoidance (Carli, Tranchina,

& Samanin, 1992), and conditioned reinforcement (Meneses, 2007) tasks, while the 5-

HT1A receptor antagonist, WAY 101405, improved learning in the Morris water maze

(Hirst et al., 2008). 5-HT1A receptor agonists and antagonists also altered performance

in the forced swim test and conditioned stress-induced ultrasonic vocalisations (Assié

et al., 2010; Lucki, Singh, & Kreiss, 1994) and altered sleep and wakefulness, as

measured by EEG and EMG (Monti & Jantos, 1992; Monti et al., 1990). Activation of

5-HT1A receptors increased impulsive responding on the five-choice serial reaction

76

time task (Carli & Samanin, 2000), while the 5-HT1A receptor antagonist, WAY

100635, supressed impulsive action (Ohmura et al., 2013).

Pharmacological manipulation of the 5-HT1B receptor subtype also affected

learning and memory as measured by a conditioned reinforcement task (Meneses,

2001, 2007), altered EEG and EMG recordings of sleep and wakefulness (Bjorvatn &

Ursin, 1994; Monti, Monti, Jantos, & Ponzoni, 1995), and affected immobility time in

the forced swim test (Dawson et al., 2006; Tatarczynska, Klodzinska, Stachowicz, &

Chojnacka-Wojcik, 2004). Therefore, it is possible that some of the cognitive and

behavioural deficits that accompany ecstasy use might be due to MDMA-produced

neuroadaptations in these receptor mechanisms.

A small number of studies have assessed the effects of repeated exposure to

MDMA on 5-HT1A and 5-HT1B receptor mechanisms. Repeated experimenter-

administered MDMA reduced 5-HT1A binding in the dorsal raphe, suggesting a down-

regulation of 5-HT1A autoreceptors, and increased 5-HT1A binding in the frontal cortex,

suggesting an up-regulation of 5-HT1A heteroreceptors (Aguirre, Ballaz, Lasheras, &

Del Rio, 1998; Aguirre, Frechilla, García-­‐Osta, Lasheras, & Del RIo, 1997; Aguirre,

Galbete, Lasheras, & Del Río, 1995). These effects were only produced following

exposure to high doses (2x20-30 mg/kg/day, 4 consecutive days); exposure to lower

doses (4x5 mg/kg/day, 2 consecutive days (McGregor et al., 2003)), or intermittent

doses (2x10mg/kg/day, every 5th day (Piper, Vu, Safain, Oliver, & Meyer, 2006)) of

MDMA failed to alter cortical or subcortical 5-HT1A densities. Repeated

administration of racemic MDMA increased 5-HT1B receptor mRNA (Kindlundh-

Högberg, Svenningsson, & Schiöth, 2006), and receptor binding densities were

increased in some brain regions, but decreased in others, after repeated MDMA

administration (McGregor et al., 2003). Repeated administration of (+) MDMA,

however, failed to produce persistent changes in 5-HT1B mRNA or 5-HT1B receptor

binding (Sexton, McEvoy, & Neumaier, 1999).

Functional evidence for these receptor changes is equivocal. Repeated

administration of MDMA attenuated the autoreceptor-mediated decrease in 5-HT

release produced by the 5-HT1A receptor agonist, F13640, in mice (Lanteri et al.,

2014). Repeated administration of MDMA did not, however, alter 8-OH-DPAT-

produced lower lip retraction or hypolocomotion, behaviours associated with 5-HT1A

autoreceptor activation (Schenk et al., 2013). On the other hand, 8-OH-DPAT-

produced hypothermia was increased after repeated MDMA administration in one

77

study (Aguirre et al., 1998) but unchanged in others (McNamara, Kelly, & Leonard,

1995; Mechan, O'Shea, Elliott, Colado, & Green, 2001; Piper et al., 2006). MDMA

pretreatment also attenuated the 8-OH-DPAT-produced 5-HT syndrome (Piper et al.,

2006) and forepaw treading (Granoff & Ashby, 2001), but had no effect on the

prosocial response (Thompson, Callaghan, Hunt, & McGregor, 2008), or the

hyperactive response (Granoff & Ashby, 2001) to 8-OH-DPAT. Differences might be

due to a number of paradigmatic variables including dosing regimen and subject

sample.

The hyperactive response to the 5-HT1B/1A receptor agonist, RU 24969, was

decreased after repeated administration of racemic MDMA (Callaway & Geyer, 1992),

but enhanced after repeated administration of the (+) MDMA isomer (McCreary,

Bankson, & Cunningham, 1999). It was suggested that this behavioural response to

RU 24969 reflected 5-HT1B receptor activation (Callaway & Geyer, 1992), but some

studies have suggested that RU 24969-produced hyperactivity is due to 5-HT1A

receptor activation (Aronsen et al., 2014; Kalkman, 1995). Repeated MDMA

administration (2x20 mg/kg/day, 4 consecutive days) failed, however, to alter

hyperactivity produced by the 5-HT1A receptor agonist, 8-OH-DPAT (Granoff &

Ashby, 2001). Therefore, the effect of MDMA exposure on the function of 5-HT1B

receptors is equivocal.

Studies on the effects of repeated exposure to MDMA have generally

administered a regimen that produces extensive, and persistent, neurotoxic effects. For

example, alterations in 5-HT1A binding, decreased tissue levels of 5-HT (Aguirre et al.,

1998) and decreased 5-HT transporter binding (Aguirre et al., 1995) were produced by

exposure to high doses (2x30mg/kg/day, 4 consecutive days) of MDMA. This high

level of exposure is rarely, if ever, experienced by ecstasy users (D. Hansen et al.,

2001; Parrott, 2005; Verheyden et al., 2003), which questions the external validity of

findings derived from these experiments (Baumann & Rothman, 2009; Cole &

Sumnall, 2003; De La Garza et al., 2007; Meyer et al., 2008).

MDMA exposure during self-administration is quite different from most

studies that employ experimenter-administered MDMA. In rats, MDMA self-

administration is initially limited, but with repeated testing intake gradually increases

for some subjects (Schenk et al., 2012). Given the differences in exposure as well as

the well documented differences between effects of contingent and non-contingent

drug administrations (Dworkin, Mirkis, & Smith, 1995; Miguéns et al., 2008), self-

78

administered MDMA might be expected to produce different effects than those seen

after experimenter-administration. Indeed, self-administered MDMA produced smaller

deficits in tissue levels of 5-HT compared to high dose experimenter-administered

MDMA (Do & Schenk, 2011; Scanzello et al., 1993; Schenk et al., 2007) even though

the total amount self-administered (165-350 mg/kg over 20-30 days of testing) was

greater than is generally administered to produce extensive neurotoxicity (20-80 mg/kg

in a single day). Additionally, intermittent or low dose exposure to MDMA was

neuroprotective against the toxic effects of subsequent high dose administrations

(Bhide et al., 2009; Piper et al., 2010).

Because of the limited amount of information concerning effects of self-

administered MDMA on brain and/or behaviour and the potential role of specific

neuroadaptations in some of the adverse effects of MDMA, this study determined the

effect of extensive MDMA self-administration on behavioural responses to 5-HT1A

and 5-HT1B receptor agonists.

Method

Subjects and procedures

Male Sprague-Dawley rats underwent catheter surgery as outlined in the

General Methods section.

MDMA self-administration

Rats were randomly assigned to self-administer either MDMA, or vehicle,

using the standard self-administration equipment outlined in the General Methods

section. Self-administration was conducted during 2 hour daily sessions, 6 days per

week. Initially, active lever responses were reinforced with MDMA (1.0 mg/kg), or

vehicle (0.1 ml) infusions according to an FR1 schedule. The vehicle control group

continued on this contingency for the remainder of the experiment. The MDMA self-

administration group continued with this contingency until a total of 90 infusions had

been self-administered, or 25 test sessions had been completed, whichever came first.

Rats that failed to self-administer 90 infusions within this 25 day cut-off period

(approximately 50%, as we have previously reported (Schenk et al., 2012)) were not

tested further. For those that met this criterion, the dose of MDMA was decreased to

0.5 mg/kg. The reinforcement schedule was then increased to FR2 for a minimum of 5

days and then FR5. Testing continued until a total intake of 350 mg/kg MDMA was

self-administered. Between 20 and 58 self-administration sessions were required to

79

reach a total intake of 350 mg/kg. Where possible, each rat in the vehicle self-

administration group was matched to a rat in the MDMA self-administration group to

ensure a comparable number of test sessions. A total of 73 rats met the initial criterion

of 90 infusions of MDMA (1.0 mg/kg/infusion) within the 25 day cut-off period. Of

these, some did not progress further due to loss of catheter patency (n=1), failure to

increase responding when the FR schedule was increased (n=12), or MDMA toxicity

(n=3). The remaining rats (n=57) completed testing and self-administered 350 mg/kg

MDMA. A total of 62 rats initiated vehicle self-administration, but 1 was removed

from the study due to an inner ear infection, leaving a total of 61 that self-administered

vehicle. Separate groups of rats that completed self-administration testing were then

randomly assigned to groups to measure the effects of either 8-OH-DPAT-produced

hyperactivity or RU 24969-produced adipsia.

Locomotor activity

Locomotor activity was assessed 2 days after the last self-administration

session. Rats were placed in the testing chamber for 30 minutes, followed by an

injection of 8-OH-DPAT (0.0, 0.03, 0.1, 0.3, 1.0 mg/kg, s.c., n=5-7 per group).

Horizontal activity counts were recorded in 5 minute intervals during the 30 minutes

prior to, and 60 minutes following, the 8-OH-DPAT injection.

Water consumption

The day following the last self-administration session, water bottles were

removed from the home cages for 24 hours. Fifteen minutes before water bottles were

reintroduced, RU 24969 (0, 0.3, 1.0, 3.0 mg/kg, s.c., n= 6-9 per group) was

administered, as previously reported (Aronsen et al., 2014). Water bottles were

weighed before, and after 30 minutes of access, to measure water consumption.

Data analysis

Effects of 8-OH-DPAT on locomotor activity were analysed by a 2 (self-

administration group) × 5 (Dose of 8-OH-DPAT) ANOVA. RU 24969-produced

adipsia was analysed with a 2 (self-administration group) × 4 (Dose of RU 24969)

ANOVA.

Results

Self-administration

The average amount of MDMA that was self-administered during the last 5

days of testing was 13.2 mg/kg/day (SEM=0.55). Figure 7.1 shows the distribution of

80

the number of rats that self-administered 350 mg/kg of MDMA as a function of test

session. Most of the rats met the criterion within 25-44 test sessions. The mean number

of test sessions required to complete testing was 35.7 (SEM=1.3). The average number

of days to complete testing reported in this study is similar to data that we have

previously reported. For example, an average of 37 +/- 2.3 days was required to self-

administer a slightly lesser total of 315 mg/kg that resulted in decreased tissue levels of

5-HT (Do & Schenk, 2011). The vehicle self-administration group was tested for an

average of 36 sessions (SEM= 1.4). These rats were matched to the MDMA self-

administration rats to minimise any confounds associated with the self-administration

procedure.

N u m b e r o f s e s s io n s

Nu

mb

er

of

rats

(n

=5

7)

2 0 -24

2 5 -29

3 0 -34

3 5 -39

4 0 -44

4 5 -49

5 0 -54

5 5 -59

0

5

1 0

1 5

Fig. 7.1 Frequency  distribution  of  the  number  of  rats  that  self-­‐administered  350  mg/kg  MDMA  as  a  function  of  test  session. 8-OH-DPAT-produced hyperactivity

Figure 7.2 shows the hyperactive response to 8-OH-DPAT after self-administration of MDMA or vehicle. ANOVA showed an effect of 8-OH-DPAT dose (F(4,47) = 27.27, p<0.01, ɳp

2= 0.70), but no effect of self-administration (F(1,47) = 0.79, p=0.38), and no interaction (F(4,47) = 0.50, p=0.50).

81

8-OH-DPAT (mg/kg)

Tota

l am

bula

tory

cou

nts

0.10

1000

2000

3000

4000VehicleMDMA

0.0 0.03 0.3 1.0

Fig. 7.2 Effect  of  MDMA  self-­‐administration  (350  mg/kg  total)  on  8-­‐OH-­‐DPAT-­‐produced  hyperactivity.  Rats  in  these  groups  met  the  criterion  of  350  mg/kg  MDMA  after  25-­‐58  test  sessions.  Symbols  represent  mean  ±  SEM.  n  =  5-­‐7  per  group.   RU 24969-produced adipsia

As we have previously shown (Aronsen et al., 2014), RU 24969 produced a

dose-dependent adipsic response (F(3,51) = 65.68, p<0.01, ɳp2= 0.79; Fig 7.3). There

was no statistically significant effect of self-administration (F(1,51) = 2.86, p=0.10)

and no statistically significant interaction (F(3,51) = 1.60, p=0.20).

RU 24969 (mg/kg)

Wat

er c

onsu

mpt

ion

(ml)

0

5

10

15 VehicleMDMA

0.0 0.3 3.01.0

Fig. 7.3 Effect  of  MDMA  self-­‐administration  (350  mg/kg  total)  on  RU  24969-­‐produced  adipsia.  Rats  in  these  groups  met  the  criterion  of  350  mg/kg  MDMA  after  20-­‐58  test  sessions.  Symbols  represent  mean  ±  SEM.  n  =  6-­‐9  per  group.

82

Discussion

MDMA self-administration failed to alter 8-OH-DPAT-produced hyperactivity,

or RU 24969-produced adipsia. It is unlikely that the MDMA exposure was

insufficient because similar or lower doses of self-administered MDMA produced

decreases in 5-HT transporter binding (Schenk et al., 2007), decreases in tissue levels

of 5-HT (Do & Schenk, 2011; Schenk et al., 2011), and behavioural deficits (Do &

Schenk, 2011). Instead, the present data suggest that 5-HT1A and 5-HT1B receptor

mechanisms are not altered by MDMA self-administration.

These findings were surprising because prolonged activation by MDMA-

produced 5-HT release might have been expected to down-regulate these receptor

subtypes. Alternatively, the decrease in MDMA-produced 5-HT release that has been

reported following MDMA self-administration (Reveron et al., 2010) might have been

expected to result in a compensatory up-regulation of these receptors. A neurotoxic

5,7-DHT lesion increased 5-HT1B receptor binding (Compan et al., 1998; Crino, Vogt,

Volicer, & Wiley, 1990; Frankfurt, Mendelson, McKittrick, & McEwen, 1993;

Manrique et al., 1998; Manrique et al., 1994; Manrique et al., 1993; Offord et al.,

1988; Weissmann et al., 1986). Furthermore, repeated agonist treatment decreased 5-

HT1B receptor binding (Pranzatelli & Razi, 1994), and behavioural responses to 5-

HT1A (De Souza, Goodwin, Green, & Heal, 1986; Hensler, 2003) and 5-HT1B (Frances

& Monier, 1991) receptor agonists.

Repeated exposure to other drugs that increase synaptic 5-HT levels altered 5-

HT1A and 5-HT1B receptors. For example, chronic treatment with the selective 5-HT

reuptake inhibitor (SSRI), fluoxetine, decreased 5-HT1B receptor binding (Duncan,

Hester, Hopper, & Franklin, 2010). It is important to note, however, that many of the

effects of SSRI treatment reflect alterations that are most likely attributed to

autoreceptor, rather than post-synaptic receptor, desensitisation. For example, repeated

treatment with fluoxetine (8 mg/kg/day, 2-3 weeks) reduced 5-HT1A mRNA in the

raphe nuclei (Le Poul et al., 2000). Higher doses also produced a decrease in 5-HT1A

receptor binding (Welner, De Montigny, Desroches, Desjardins, & Suranyi-­‐Cadotte,

1989) and 8-OH-DPAT stimulated [35S]GTPγS binding (Castro et al., 2003) in the

dorsal raphe. Repeated exposure to MDMA failed to alter a number of 5-HT1A

autoreceptor mediated behavioural or neurochemical responses (Schenk et al., 2013),

suggesting differences between effects of these two classes of drugs. Repeated

administrations of cocaine increased 5-HT1B receptor binding (Przegaliński, Czepiel,

83

Nowak, Dlaboga, & Filip, 2003) and 5-HT1B mRNA (Hoplight, Vincow, & Neumaier,

2007). Cocaine self-administration also increased the behavioural and physiological

responses to 5-HT1A and 5-HT1B receptor agonists (O'Dell, Manzardo, Polis, Stouffer,

& Parsons, 2006).

The present data do not rule out the possibility that repeated ecstasy use leads

to cognitive and behavioural deficits via dysregulation of these receptor subtypes, but

our results suggest that other 5-HT receptors are more likely to make important

contributions. One potential candidate is the 5-HT2A receptor, because it has also been

implicated in impulsivity (Cunningham & Anastasio, 2014) , sleep (Sharpley, Elliott,

Attenburrow, & Cowen, 1994) and memory (Dhonnchadha & Cunningham, 2008;

Howell & Cunningham, 2015), behaviours that are impacted by regular ecstasy use.

MDMA exposure increased 5-HT2A receptor binding (Benningfield & Cowan, 2013;

Urban et al., 2012) and behavioural responses to the 5-HT2A/2C receptor agonist, DOI

(Biezonski, Courtemanche, Hong, Piper, & Meyer, 2009). Additional studies assessing

the impact of MDMA self-administration on this receptor mechanism is warranted.

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General Discussion

Summary

MDMA is widely used in the form of the street drug, ecstasy. Regular use of

ecstasy has been associated with a number of behavioural and neurochemical deficits,

and some of these deficits likely contribute to further, problematic drug taking. While

most drugs of abuse primarily enhance dopamine neurotransmission, MDMA

preferentially releases 5-HT. This 5-HT release has been hypothesised to inhibit the

dopaminergic response to MDMA, thus inhibiting the reinforcing efficacy of MDMA.

However, with repeated exposure to MDMA, the 5-HTergic response is attenuated,

disinhibiting the dopaminergic response and making MDMA similar to other drugs of

abuse. The mechanism for this 5-HTergic inhibition of dopamine is not known, but

one possibility is that activation of specific 5-HTergic receptors, via MDMA-produced

5-HT release, alters the dopaminergic response to MDMA. Of the 14 different 5-HT

receptors, the 5-HT1A and 5-HT1B receptors were investigated because of a

documented role in regulating basal and drug-produced dopamine release, as well as

behaviours associated with ecstasy use. The purpose of this thesis was to test the role

of these receptors in the self-administration of MDMA in rats, and to document any

changes in these receptor populations produced by MDMA.

Firstly, appropriate behavioural assays for 5-HT1A and 5-HT1B receptor

activation needed to be identified. Latent inhibition, measured using the conditioned

taste aversion paradigm, was chosen as a behavioural response to 5-HT1B receptor

activation, but this response was confounded by the adipsic response to the 5-HT1B/1A

receptor agonist, RU 24969. After further testing, I found that this adipsic response to

RU 24969 was dose-dependent, and blocked by a 5-HT1B, but not a 5-HT1A, receptor

antagonist. Thus, the adipsic response to RU 24969 was chosen as a behavioural

measure of 5-HT1B receptor activation. In contrast, the hyperactive response to RU

24969 was blocked by a 5-HT1A, but not a 5-HT1B, receptor antagonist. A similar result

was obtained with the more selective and well characterised 5-HT1A receptor agonist,

8-OH-DPAT, thus the locomotor response to 8-OH-DPAT was chosen as a

behavioural response to 5-HT1A receptor activation.

To test whether 5-HT1A and/or 5-HT1B receptors regulated MDMA self-

administration I attempted to alter the activity of these receptors and measure the

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impact on the acquisition of MDMA self-administration. To this end, rats were

repeatedly administered a high dose of the 5-HT1B/1A receptor agonist, RU 24969,

before commencing MDMA self-administration. The pretreatment down-regulated 5-

HT1A and 5-HT1B receptors, and greatly facilitated the acquisition of MDMA self-

administration. Because drug produced dopamine release is inhibited by activation of

5-HT1A receptors, but enhanced by 5-HT1B receptors, the impact of RU 24969

pretreatment on acquisition of MDMA self-administration was hypothesised to be

associated with the down-regulation of 5-HT1A receptors.

The role of 5-HT1A and 5-HT1B receptors in the acquisition of MDMA self-

administration was further tested by investigating the relationship between basal

responses to receptor activation and latency to acquire MDMA self-administration.

Based on the role of these receptors in regulating the dopaminergic response to other

drugs of abuse, and the facilitated acquisition of MDMA self-administration after

repeated exposure to RU 24969, it was expected that behavioural responses to

activation of these receptors would predict the latency to acquire MDMA self-

administration. This hypothesis was not supported in any of the studies. Furthermore,

because an inhibitory role of 5-HT1A receptor activation in the acquisition of MDMA

self-administration was hypothesised, I investigated the effect of 5-HT1A receptor

antagonist treatment during the acquisition phase. Again, results did not support the

hypothesis, in fact, the results suggested that 5-HT1A receptor blockade inhibited

MDMA self-administration.

Acquisition studies had returned mainly negative results, but there was still

reason to believe that 5-HT1A and/or 5-HT1B receptors regulated the self-administration

of MDMA. Therefore, behavioural responses to 5-HT1A or 5-HT1B receptor activation

were measured after the self-administration of a high dose of MDMA. It was expected

that prolonged exposure to MDMA would alter behavioural responses to agonist

administration, but again this hypothesis was not supported. Although these studies do

not rule out the possibility of 5-HT1A or 5-HT1B receptor neuroadaptations in response

to MDMA self-administration, they do suggest that other 5-HT receptors are more

likely to make important contributions.

Synthesis of results

Overall, the data presented in this thesis are difficult to reconcile. On the one

hand, there is a sound theoretical basis to expect that 5-HT1A and 5-HT1B receptors

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would modulate the reinforcing efficacy of MDMA, and that these receptor

mechanisms would be altered by prolonged exposure to MDMA. Furthermore, the RU

24969 pretreatment, that down-regulated 5-HT1A and 5-HT1B receptors, also facilitated

the acquisition of MDMA self-administration. On the other hand, behavioural

responses to 5-HT1A and 5-HT1B receptor activation did not predict the acquisition of

MDMA self-administration, acquisition was blocked by the 5-HT1A receptor

antagonist, WAY 100635, and there were no changes in dose response curves for 5-

HT1A or 5-HT1B mediated responses after the self-administration of a high dose of

MDMA.

Together, the most likely explanation for the results of the studies comprising

this thesis is that 5-HT1A and 5-HT1B receptors have a limited role in the self-

administration of MDMA. If true, this conclusion would suggest that the facilitated

self-administration produced by RU 29496 pretreatment was due to some non-specific

effect. To test this possibility, it would be important to co-administer a 5-HT1A or 5-

HT1B receptor antagonist with RU 24969 during pretreatment and test for latency to

acquire MDMA self-administration.

Limitations

It is possible that the conclusions made in this thesis were skewed by the

behavioural measures used. Although it was demonstrated that RU 24969-produced

adipsia and 8-OH-DPAT-produced locomotor activity are measures of 5-HT1B and 5-

HT1A receptor activation, respectively, there is no consensus on what population of 5-

HT1B or 5-HT1A receptors produce these effects. Systemic administration of 8-OH-

DPAT produced dose-dependent hyperactivity, but this locomotor response is the net

result of global 5-HT1A activation. Specific 5-HT1A populations alter locomotor

activity in different ways, for example local injections of 8-OH-DPAT in the PFC did

not alter locomotor activity (Solati et al., 2011), while administration in the NAc

decreased locomotor activity (Hillegaart, Ahlenius, & Larsson, 1991; Plaznik et al.,

1994). Similarly, the population of 5-HT1B receptors responsible for the adipsic

response to RU 24969 is not known. One study showed that local infusion of RU

24969, or the more selective 5-HT1B receptor agonist, CP 93129, in the NAc reduced

responding for water (P. J. Fletcher & Korth, 1999a), but it is not clear what other

populations of 5-HT1B receptors might also influence this behavioural response. Thus,

care needs to be taken when interpreting these behavioural data. It is possible, for

87

example, that MDMA self-administration did alter some 5-HT1A and/or 5-HT1B

receptor populations, but not those that impact the locomotor response to 8-OH-DPAT

or the adipsic response to RU 24969.

Extensive study would be required to address the possibility that the negative

results found in this thesis were due to the choice of behavioural responses. Because of

the time, rats, and drugs required, it was not possible to investigate further for this

thesis, but our lab has started to probe this possibility in further detail. The logical first

step is to directly investigate the effect of MDMA self-administration on 5-HT1A and

5-HT1B receptor binding. There is a widely used and well characterised 5-HT1A

antibody (Abbas et al., 2007; Kia et al., 1996; Say, Machaalani, & Waters, 2007;

Tachibana, Endoh, Fujiwara, & Nawa, 2005), allowing for an immunohistochemistry

investigation, but 5-HT1B receptors are best mapped using a radioactively labelled

ligand (Domenech, Beleta, & Palacios, 1997; Lindhe et al., 2011). Our lab is currently

conducting 5-HT1A immunohistochemistry on tissue from rats that have extensive

MDMA self-administration history. Such an approach allows for a detailed, region

specific, analysis of the effect of MDMA self-administration. Similar data could be

obtained to determine the effect of RU 24969 pretreatment. Receptor populations that

are similarly affected by both manipulations might underlie the development of

MDMA as an efficacious reinforcer. Thus, local drug administrations in these areas

could be used, first to obtain a behavioural response to predict the acquisition of

MDMA self-administration, then for pharmacological treatments to reduce MDMA

self-administration.

Validity of MDMA doses

The self-administration paradigm was used in this thesis because it likely

produces neuroadaptations similar to those produced by ecstasy use in humans. It is

important to note that, when compared across species, the MDMA doses self-

administered in this thesis were of relevance to human users. The issue of interspecies

scaling is based on the fact that, in general, smaller animals have relatively larger

organs and a shorter blood circulation time, and so will metabolise drug faster

(Mordenti & Chappell, 1989). Therefore, as long as there are no species-specific

mechanisms of drug metabolism, smaller animals require larger doses in order for

effects to be comparable to those produced in larger animals. Most recreational users

consume 1-2 tablets per ecstasy-taking session (Parrott, 2005; Verheyden et al., 2003),

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and although the contents of ecstasy tablets procured ‘on the street’ vary widely,

median MDMA content per tablet has been shown to be around 70-80 mg (Vogels et

al., 2009). Therefore, a 70kg user is consuming approximately 1-2 mg/kg MDMA in

recreational settings. Interspecies scaling can help to determine the doses that should

be administered in animal studies to best mimic the effects of such doses in

recreational users.

As a starting point for investigating drugs across species the USA Food and

Drug Administration (FDA) suggest that drug doses should be scaled across species

based on the body weight and surface area of these species (Food and Drug

Administration, 2005). The FDA recommendation is that the effects of a 1.0 mg/kg

dose in a human are roughly comparable with the effects of a 6.2 mg/kg dose in a rat.

The FDA scaling suggestions are not drug-specific and are meant merely as a

guideline for determining safe initial doses in clinical trials.

With specific reference to MDMA, some researchers have used the following

algorithm to scale doses between species:

DHuman = DAnimal (WHuman / WAnimal) k (Equation 1)

where D is the drug dose in mg, W is weight in kg, and k is an estimated value that

reflects the logarithmic relationship between bodyweight and metabolic rate (that is,

the slope of the curve fitted to the log transforms of empirical values for weight and

drug clearance times of different species). This process, in which doses are determined

by transforming bodyweight to a different physiological variable through a power

function, is called ‘allometric scaling’ (Mordenti & Chappell, 1989). The precise value

of the scaling factor, k, has been seriously contended in the literature, with suggestions

ranging between 0.67 and 0.77 (Food and Drug Administration, 2005; Mordenti &

Chappell, 1989; Travis & White, 1988; Watanabe, Bois, & Zeise, 1992). In studies

using MDMA, a k value of 0.7 has been adopted (McCann & Ricaurte, 2001; Ricaurte,

Yuan, & McCann, 2000). Based on Equation 1, with k set at 0.7, a 1.0 mg/kg dose in a

70kg human would be equivalent to a 5.0 mg/kg dose in a 330g rat. It should be noted

that this suggested dose could vary from 3.4 mg/kg to 5.9 mg/kg if the highest or

lowest suggested k value is used, respectively.

Vollenweider, Jones, and Baggott (2001) have suggested that allometric scaling

is not relevant to MDMA because there is evidence for species differences in MDMA

pharmacokinetics, and because MDMA has active metabolites that may contribute to

the drug effect. Instead, they suggest that pharmacokinetic data (e.g. area under the

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curve (AUC) of MDMA plasma levels) should be compared between species to

determine similar doses. Based on equation 1, McCann and Ricaurte (2001) claim that

a 20 mg/kg dose of MDMA in a 220g rat is comparable to a 1.4 mg/kg dose in a

human, however AUC of MDMA plasma concentrations in humans after

approximately 1.8 mg/kg was 70% lower than the AUC in rats after 20 mg/kg

(Vollenweider et al., 2001). Although comparing pharmacokinetic data can account for

some potential flaws with allometric scaling, comparisons can only be made with

empirical data, so finding similar doses across species becomes a ‘trial and error’ type

task.

Both allometric scaling and comparisons of pharmacokinetic data (AUC)

suggest that a human dose of approximately 1 mg/kg is comparable to a rat dose of

roughly 5 mg/kg (De La Torre et al., 2000; Fitzgerald, Blanke, & Poklis, 1990). Thus,

during initial self-administration sessions, in which rats self-administer less than 5

infusions per session, rats are consuming less MDMA than a human user might be

expected to use recreationally. As intake increases, rats will self-administer 5-10

mg/kg per session, which roughly scales to the human recreational dose. The total

intake of 350 mg/kg MDMA used in this thesis is comparable to that of a heavy

ecstasy user, after roughly 70 recreational doses. Most studies have shown cognitive or

behavioural deficits in ecstasy users to be present at levels of total intake around or

below 70 doses (Booij et al., 2014; A. K. Davis & Rosenberg, 2014; McCann, Mertl, et

al., 1999; Wagner et al., 2013). Therefore, the doses of MDMA self-administered by

rats in this thesis are relevant to human ecstasy users, particularly heavy users.

Key findings and future directions

This thesis made a number of novel and important findings. Firstly,

characterising the adipsic and hyperactive responses to RU 24969 as 5-HT1B and 5-

HT1A receptor mediated, respectively, was important for clarifying previous findings

and facilitating further research. Earlier studies had not clearly shown the mechanism

by which RU 24969 produced hyperactivity, and some had interpreted the hyperactive

response as a behavioural measure of 5-HT1B receptor activation (Callaway & Geyer,

1992). The results from this thesis clearly show a role of 5-HT1A, but not 5-HT1B

receptors in RU 24969-produced hyperactivity. Furthermore, this thesis provides a

straightforward behavioural assay for 5-HT1B receptor activation, and this behavioural

measure might be useful in preclinical tests. As outlined above, further study of the

90

population of 5-HT1B receptors that produce the the adipsic response to RU 24969

would make this behavioural assay more useful.

As one reviewer pointed out, the facilitation of MDMA self-administration

produced by RU 24969 pretreatment is a ‘novel and important’ finding for the

addiction field, although more work needs to be done to understand this effect. I would

strongly encourage further investigation of the effects of RU 24969 pretreatment on 5-

HTergic systems so that the mechanism by which this pretreatment facilitated MDMA

self-administration can be elucidated. Another novel finding made in this thesis was

that MDMA self-administration had no effect on behavioural responses to 5-HT1A or

5-HT1B activation. Again, a reviewer commented that these results are interesting and

important, even though the results were negative.

The studies in this thesis were based on the theory that a decreased 5-HTergic

response to MDMA after repeated exposure could enhance the reinforcing effects of

MDMA via altered activation of 5-HT receptors that regulate dopamine

neurotransmission. Although this thesis suggests that 5-HT1A and 5-HT1B receptors

likely play a limited role in the enhanced reinforcing efficacy of MDMA after repeated

exposure, the theoretical basis for these studies is still sound. Thus, it is possible that

there are other 5-HT receptors that regulate the reinforcing efficacy of MDMA and

that also underlie cognitive and behavioural deficits following repeated exposure. Two

5-HT receptors that have been shown to regulate dopaminergic neurotransmission are

the 5-HT2A and 5-HT2C receptors.

5-HT2C receptors are well localised to mediate the dopaminergic responses to

drugs of abuse, with high levels of 5-HT2C receptors reported in dopamine terminal

areas of the PFC, striatum, and NAcc, and in the VTA (Bubar & Cunningham, 2006;

Clemett, Punhani, Duxon, Blackburn, & Fone, 2000; Di Matteo, De Blasi, Di Giulio,

& Esposito, 2001; Eberle-­‐Wang, Mikeladze, Uryu, & Chesselet, 1997; Ji et al., 2006).

5-HT2C receptor agonists inhibited, while 5-HT2C receptor antagonists enhanced, the

firing rate of VTA dopamine neurons, and extracellular dopamine levels in the nucleus

accumbens and PFC (Alex, Yavanian, McFarlane, Pluto, & Pehek, 2005; Di Matteo et

al., 2001).

The 5-HT2C/2B receptor antagonist, SB 206553, and the more selective 5-HT2C

receptor antagonist, SB 242084, both potentiated the cocaine-produced increase in

extracellular dopamine in the nucleus accumbens and striatum (Navailles, De

Deurwaerdere, Porras, & Spampinato, 2004).The 5-HT2C receptor agonist, Ro 60-175,

91

inhibited the self-administration of cocaine, ethanol, and nicotine, an effect that was

reversed by the 5-HT2C receptor antagonist, SB 242084 (P. J. Fletcher, Chintoh,

Sinyard, & Higgins, 2004; P. J. Fletcher, Rizos, Sinyard, Tampakeras, & Higgins,

2007; Grottick, Corrigall, & Higgins, 2001; Grottick, Fletcher, & Higgins, 2000;

Tomkins et al., 2002). Mice that lack the 5-HT2C gene reached higher breakpoints in a

progressive ratio paradigm reinforced by cocaine, and also showed enhanced levels of

cocaine-induced dopamine release in the NAcc (Rocha et al., 2002).

These findings are consistent with the idea that activation of 5-HT2C receptors

is inhibitory to, while blockade of 5-HT2C receptors facilitates, the dopaminergic

response to drugs of abuse. As such, it is possible that a down-regulation of 5-HT2C

receptors, in response to repeated exposure to MDMA, underlies the development of

MDMA as an efficacious reinforcer

Unfortunately, there is no clear evidence for 5-HT2C receptor down-regulation

in response to MDMA exposure. On the one hand, male ecstasy users showed blunted

neuroendocrine responses to the 5-HT2/1A receptor agonist, m-CPP, compared to

MDMA-naïve controls (McCann, Eligulashvili, Mertl, Murphy, & Ricaurte, 1999),

and repeated administration of MDMA decreased 5-HT2C receptor protein levels in the

hippocampus of young-adult rats (García-Cabrerizo & García-Fuster, 2015),

suggesting a possible down-regulation of 5-HT2C receptors after MDMA exposure. On

the other hand, repeated exposure to MDMA enhanced the inhibition of MDMA-

produced hyperlocomotion by the 5-HT2C receptor agonist, MK 212 (Ramos, Goni-

Allo, & Aguirre, 2005) and increased sensitivity to the 5-HT2/1A receptor agonist, m-

CPP (Taffe et al., 2002). Furthermore, repeated exposure to MDMA increased 5-HT2C

mRNA in cortex and hypothalamus (Kindlundh-Högberg et al., 2006). Further still,

some animal studies have failed to show any effect of MDMA exposure on

neuroendocrine or behavioural responses to m-CPP (Bull et al., 2003; Jones, Brennan,

Colussi-Mas, & Schenk, 2010).

It is entirely possible that repeated exposure to MDMA in the self-

administration paradigm would down-regulate 5-HT2C receptors, but so far there is

limited evidence to suggest this would be the case. Significantly more research is

required to determine the effects of MDMA self-administration on 5-HT2C receptor

mechanisms. On the other hand, there is substantial evidence to suggest that

neuroadaptations in 5-HT2A receptor mechanisms might underlie the development of

MDMA as an efficacious reinforcer in the self-administration paradigm.

92

5-HT2A receptors are strongly expressed as excitatory 5-HTergic receptors on

non-5-HTergic cells in the PFC (Eison & Mullins, 1995), where their activation has

been shown to increase dopamine activity in the VTA (Bortolozzi, Díaz-Mataix,

Scorza, Celada, & Artigas, 2005). This increased mesocorticolimbic dopamine release

is a product of increased glutamatergic activity in projections from the PFC to the

VTA (Aghajanian & Marek, 1999; Pehek, Nocjar, Roth, Byrd, & Mabrouk, 2005). The

5-HT2A/2C receptor antagonist, ketanserin, attenuated the dopaminergic response to

MDMA in the striatum (Nash, 1990), and a similar effect was produced by local

administration of the selective 5-HT2A receptor antagonist, M100907, in the striatum

(Schmidt, Sullivan, & Fedayal, 1994). On the other hand the non-selective 5-HT2

receptor agonists, DOI and 5-MeODMT, both enhanced the dopaminergic response to

MDMA in the striatum (Gudelsky, Yamamoto, & Nash, 1994). These data suggest that

activation of the 5-HT2A receptor via MDMA-induced 5-HT release would enhance the

reinforcing efficacy of MDMA.

Compellingly, repeated MDMA was associated with increased 5-HT2A receptor

binding (Benningfield & Cowan, 2013; Urban et al., 2012)(but see McGregor et al.

(2003)), suggesting that MDMA self-administration might also up-regulate 5-HT2A

receptors. Thus, with repeated exposure to MDMA, enhanced activation of 5-HT2A

receptors could underlie the development of MDMA as an efficacious reinforcer. This

hypothesis is in agreement with the finding that a neurotoxic 5,7-DHT lesion, which

also facilitated the acquisition of MDMA self-administration (Bradbury et al., 2014),

produced an increase in 5-HT2A receptor binding density in mice (Heal, Philpot,

Molyneux, & Metz, 1985).

An up-regulation of 5-HT2A receptors might also underlie the increased

impulsivity produced by repeated exposure to MDMA. The 5-HT2A/2C receptor agonist,

DOI, increased premature responding on the 5CSRTT (Koskinen, Haapalinna, & Sirvi,

2003; Koskinen & Sirviö, 2001), and this effect was blocked by the 5-HT2A/2C receptor

antagonist, ketanserin (Koskinen, Ruotsalainen, Puumala, et al., 2000; Koskinen,

Ruotsalainen, & Sirviö, 2000), while ketanserin (P. J. Fletcher, Tampakeras, Sinyard,

& Higgins, 2007; Passetti, Dalley, & Robbins, 2003; Ruotsalainen et al., 1997; Talpos,

Wilkinson, & Robbins, 2006) and the more selective 5-HT2A receptor antagonist,

M100907 (P. J. Fletcher, Tampakeras, et al., 2007; Winstanley, Theobald, Dalley,

Glennon, & Robbins, 2004), decreased premature responses on the 5CSRTT, or the

93

similar 1CSRTT (Anastasio et al., 2011). Increased impulsivity, due to an up-

regulation of 5-HT2A receptors after repeated exposure to MDMA, would be expected

to facilitate drug taking, and as such could underlie the development of an MDMA

SUD (Schenk & Aronsen, 2015).

The role of the 5-HT2A receptor in MDMA self-administration has not been

studied. Some studies have shown no effect of regular, repeated MDMA

administration on the head-twitch or locomotor responses to the 5-HT2A/2C receptor

agonist, DOI (Granoff & Ashby Jr, 1998), or the behavioural response to the non-

selective 5-HT2 receptor agonist, mCPP (Jones et al., 2010). On the other hand, an

intermittent dosing regimen of MDMA increased the head-twitch responses to DOI

(Biezonski et al., 2009), suggesting that adaptations in 5-HT2A receptors may be

dependent on dosing regimen. Ecstasy use is typically intermittent, and 5-HT2A

binding was increased in human ecstasy users, compared to naïve controls, with

increased exposure to MDMA associated with increased 5-HT2A binding density (Di

Iorio et al., 2012; Urban et al., 2012). Thus, it is possible that an up-regulation of 5-

HT2A receptors would be evident after MDMA self-administration.

It would be interesting to selectively up-regulate 5-HT2A receptors and test for

latency to acquire MDMA self-administration. An up-regulation may be achieved by

repeatedly administering the selective 5-HT2A receptor antagonist, M100907 (Minabe,

Hashimoto, Watanabe, & Ashby, 2001). Behavioural assessment is difficult, however,

because of the lack of selective 5-HT2A receptor agonists. Ideally, a behavioural

response to the selective antagonist, M100907, would be determined. There are some

reports that M100907 enhanced the inhibition of a startle response in the pre-pulse

inhibition paradigm (Padich, McCloskey, & Kehne, 1996; Zhang, Engel, Jackson,

Johansson, & Svensson, 1997), although more parametric work for this behavioural

response is required (Geyer, Krebs-Thomson, & Varty, 1999; Varty, Bakshi, & Geyer,

1999)

Conclusion

Repeated exposure to MDMA enhances the reinforcing efficacy of MDMA. It

is possible that this increased reinforcement is due to adaptations in 5-HT receptors

that regulate dopaminergic responses to MDMA. This thesis showed that 5-HT1A and

5-HT1B receptors likely play a limited role in the self-administration of MDMA, and

thus likely do not explain the enhanced reinforcing efficacy of MDMA after repeated

94

exposure. Future research should consider the role of the 5-HT2A receptor in

neuroadaptations that might underlie the self-administration of MDMA.

95

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Appendix A: Publication details and permissions

Aronsen, Webster, & Schenk (2014). RU 24969-produced adipsia and

hyperlocomotion: Differential role of 5HT1A and 5HT1B receptor mechanisms.

Pharmacology, Biochemistry and Behavior, 124, 1-4. DOI

10.1016/j.pbb.2014.05.008

Licensed content publisher: Elsevier

License Number: 3805061346001

License date: Feb 09, 2016

Schenk & Aronsen (2015). Contribution of Impulsivity and Serotonin Receptor

Neuroadaptations to the Development of an MDMA (“ecstasy”) Substance Use

Disorder, in Current Topics in Behavioral Neuroscience: The Misuse of Licit

and Illicit Drugs in Psychopharmacology Susan Schenk, Suzanne Nielson,

Raimondo Bruno (Eds), Springer. DOI 10.1007/7854_2015_421

Licensed content publisher: Springer

License Number: 3805070143872

License date: Feb 09, 2016

Aronsen, Bukholt, & Schenk (2016). Repeated administration of the 5-HT1B/1A agonist,

RU 24969, facilitates the acquisition of MDMA self-administration: Role of 5-

HT1A and 5-HT1B receptor mechanisms. Psychopharmacology, 233 (8), 1339-

1347. DOI 10.1007/s00213-016-4225-x

Licensed content publisher: Springer

License Number: 3805490889197

License date: Feb 10, 2016

Aronsen & Schenk (2016). MDMA self-administration fails to alter the behavioral

response to 5-HT1A and 5-HT1B agonists. Psychopharmacology, 233 (7), 1323-

1330. DOI 10.1007/s00213-016-4226-9

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Licensed content publisher: Springer

License Number: 3805491012086

License date: Feb 10, 2016